Method of screening TGF-beta-inhibiting substances

ABSTRACT

A method for screening substances that inhibit binding between a TAK1 polypeptide and a TAB1 polypeptide, which comprises contacting the TAB1 polypeptide to the TAK1 polypeptide and a test sample and then detecting or determining the TAK1 polypeptide that is bound to the TAB1 polypeptide.

TECHNICAL FIELD

[0001] The present invention relates to a method for screeningsubstances that inhibit binding between TAK1 and TAB1. The presentinvention also relates to a method for screening substances that inhibitthe signal transduction of transforming growth factor-β (TGF-β). Thepresent invention further relates to substances and uses thereofobtainable by the method for screening substances that inhibit bindingbetween TAK1 and TAB1.

BACKGROUND ART

[0002] Transforming growth factorβ (TGF-β) is a multilfunctional factorthat controls various aspects of cell functions. As one such function,TGF-β is responsible for the repair and regeneration of tissuesassociated with various injuries (Border, W. A. & Noble, N. A., The NewEngland Journal of Medicine (1994) 331, 1286-1292).

[0003] An abnormal production of TGF-β in chronic injuries can sometimesdisturb balances in the repair and regeneration of tissues resulting inpathological fibrosis. As a pathological condition in which the balanceof TGF-β production has been disturbed, hepatic fibrosis is known. Ithas been elucidated that TGF-β acts as a main causative agent offibrosis of various organs such as the liver, by enhancing theproduction of extracellular matrix protein that can cause fibrosis,inhibiting the synthesis of proteolytic enzymes of extracellular matrix,and by inducing substances that inhibit proteolytic enzymes ofextracellular matrix (Border, W. A. & Noble, N. A. , The New EnglandJournal of Medicine (1994) 331, 1286-1292).

[0004] Other known functions of TGF-β include the activity. ofinhibiting cellular growth (Moses, H. L. et al., Cell (1990) 63,245-247), the activity of migrating monocytes (Wahl, S. M. et al., Proc.Natl. Acad. Sci. U.S.A. (1987) 84, 5788-5792), the activity of inducingbiologically active substances (Wahl, S. M. et al., Proc. Natl. Acad.Sci. U.S.A. (1987) 84, 5788-5792), the activity of facilitating thedeposition of amyloid β protein (Wyss-Coray, T. et al., Nature (1997)389, 603-606), and the like.

[0005] TGF-β transduces its signals through heteromer complexes of typeI and type II TGF-β receptors and transmembrane proteins containing theserine- and threonine-specific kinase domains at the side of cytoplasm(Wrana, J. L. et al., Nature (1994) 370, 341; Kingsley, D. M. et al.,Genes Dev. (1994) 8, 133). However, much of the mechanism of signalingdownward from the TGF-β receptor into the cell on the molecular levelremains to be elucidated.

[0006] As a series of systems involved in the signal transduction of theTGF-β superfamily, mitogen-activated protein kinase (MAPK) is known.

[0007] The MAPK system is a conserved eukaryotic signaling system thatconverts signals of a receptor into various functions. The MAPK systemcontains three types of protein kinases, i.e. mitogen-activated proteinkinase kinase kinase (MAPKKK), mitogen-activated protein kinase kinase(MAPKK), and mitogen-activated protein kinase (MAPK). MAPK is activatedthrough phosphorylation by MAPKK. MAPKK is activated throughphosphorylation by MAPKKK (Nishida, E. et al., Trends Biochem. Sci.(1993) 18, 128; Blumer, K. J. et al., Trends Biochem. Sci. (1993) 19,236; David R. J. et al., Trends Biochem. Sci. (1993) 19, 470; Marchall,C. J. et al., Cell (1995) 80, 179).

[0008] TAK1 (TGF-β-activated kinase 1), that is a member of the MAPKKKfamily that functions in the signaling system of biologically activesubstances and that belongs to the TGF-β superfamily, was identified byYamaguchi, K. et al. (Yamaguchi, K. et al., Science (1995) 270, 2008).

[0009] TAB1 (TAK1 binding protein 1), a protein involved in thesignaling system of TGF-β that binds to and activates TAK1, wasidentified by Shibuya, H. et al. (Shibuya, H. et al., Science (1996)272, 1179-1182).

[0010] Although TAB1 transduces the signal of TGF-β by binding to TAK1and activating TAK1 kinase activity, no attempts have been made so farto search for substances that inhibit binding between TAK1 and TAB1 inorder to suppress or activate signal transduction of TGF-β by focusingon the binding between TAK1 and TAB1.

DISCLOSURE OF THE INVENTION

[0011] The present invention is intended to provide a method forscreening substances that inhibit binding between TAK1 and TAB1. Thepresent invention is also intended to provide a method for screeningsubstances that suppress or activate the signal transduction of TGF-β.The present invention further is intended to provide substances that areobtainable by a method for screening substances that inhibit bindingbetween TAK1 and TAB1.

[0012] Thus, the present invention provides (1) a method for screeningsubstances that inhibit binding between a TAK1 polypeptide and a TAB1polypeptide, which method comprises contacting the TAB1 polypeptide tothe TAK1 polypeptide and a test sample and then detecting or determiningthe TAK1 polypeptide that is bound to the TAB1 polypeptide. Preferably,the TAB1 polypeptide is a TAB1 polypeptide that has been bound to asupport. A preferred support is beads or a plate. In another preferredembodiment, the contact between a TAK1 polypeptide, a TAB1 polypeptideand a test sample is carried out in a homogeneous system.

[0013] The present invention also provides (2) a method for screeningsubstances that inhibit binding between a TAK1 polypeptide and a TAB1polypeptide, which method comprises contacting the TAK1 polypeptide tothe TAB1 polypeptide and a test sample, and then detecting ordetermining the TAB1 polypeptide that is bound to the TAK1 polypeptide.Preferably, the TAK1 polypeptide is a TAK1 polypeptide that has beenbound to a support. A preferred support is beads or a plate. In anotherpreferred embodiment, the contact between a TAK1 polypeptide, a TAB1polypeptide and a test sample is carried out in a homogeneous system.

[0014] The present invention also provides (3) a screening methoddescribed in the above (1) and (2), which method comprises using a TAB1polypeptide having an amino acid sequence comprising Met at amino acidposition 1 to Pro at amino acid position 504 of the amino acid sequenceas set forth in SEQ ID NO: 2, or having an amino acid sequence modifiedby the substitution, deletion and/or addition of one or a plurality ofamino acid residues of the amino acid sequence as set forth in SEQ IDNO: 2 and maintaining the biological activity of the TAB1 polypeptide;and/or

[0015] a TAK1 polypeptide having an amino acid sequence comprising Metat amino acid position 1 to Ser at amino acid position 579 of the aminoacid sequence as set forth in SEQ ID NO: 4, or having an amino acidsequence modified by the substitution, deletion and/or addition of oneor a plurality of amino acid residues of the amino acid sequence as setforth in SEQ ID NO: 4 and maintaining the biological activity of theTAK1 polypeptide.

[0016] The present invention also provides (4) a screening methoddescribed in the above (1) to (3), which comprises using a TAK1polypeptide fused to another peptide or polypeptide and/or a TAB1polypeptide fused to another peptide or polypeptide.

[0017] The present invention also provides (5) a method for screeningsubstances that inhibit binding between a TAK1 polypeptide and a TAB1polypeptide, which method comprises contacting the TAB1 polypeptide tothe labeled TAK1 polypeptide and a test sample, and then detecting ordetermining the labeled TAK1 polypeptide that is bound to the TAB1polypeptide. Preferably, the TAB1 polypeptide is a TAB1 polypeptide thathas been bound to a support. A preferred support is beads or a plate. Inanother preferred embodiment, the contact between a TAK1 polypeptide, aTAB1 polypeptide and a test sample is carried out in a homogeneoussystem.

[0018] The present invention also provides (6) a method for screeningsubstances that inhibit binding between a TAK1 polypeptide and a TAB1polypeptide, which method comprises contacting the TAK1 polypeptide tothe labeled TAB1 polypeptide and a test sample, and then detecting ordetermining the labeled TAB1 polypeptide that is bound to the TAK1polypeptide. Preferably, the TAK1 polypeptide is a TAK1 polypeptide thathas been bound to a support. A preferred support is beads or a plate. Inanother preferred embodiment, the contact between a TAK1 polypeptide, aTAB1 polypeptide and a test sample is carried out in a homogeneoussystem.

[0019] The present invention also provides (7) a screening methoddescribed in the above (5) and (6), which method comprises using a TAB1polypeptide having an amino acid sequence comprising Met at amino acidposition 1 to Pro at amino acid position 504 of the amino acid sequenceas set forth in SEQ ID NO: 2 or having an amino acid sequence modifiedby the substitution, deletion and/or addition of one or a plurality ofamino acid residues of the amino acid sequence as set forth in SEQ IDNO: 2 and maintaining the biological activity of the TAB1 polypeptide;and/or

[0020] a TAK1 polypeptide having an amino acid sequence comprising Metat amino acid position 1 to Ser at amino acid position 579 of the aminoacid sequence as set forth in SEQ ID NO: 4 or having an amino acidsequence modified by the substitution, deletion and/or addition of oneor a plurality of amino acid residues of the amino acid sequence as setforth in SEQ ID NO: 4 and maintaining the biological activity of theTAK1 polypeptide.

[0021] The present invention also provides (8) a screening methoddescribed in the above (5) to (7), which method comprises using a TAK1polypeptide fused to another peptide or polypeptide and/or a TAB1polypeptide fused to another peptide or polypeptide. Preferably, saidlabeled TAK1 polypeptide or said labeled TAB1 polypeptide is a TAK1polypeptide or a TAB1 polypeptide that is labeled with a radioisotope,an enzyme or a fluorescent substance.

[0022] The present invention also provides (9) a method for screeningsubstances that inhibit binding between a TAK1 polypeptide and a TAB1polypeptide, which method comprises contacting the TAB1 polypeptide tothe TAK1 polypeptide and a test sample, and then detecting ordetermining the TAK1 polypeptide that is bound to the TAB1 polypeptideby a primary antibody against the TAK1 polypeptide. Preferably, the TAB1polypeptide is a TAB1 polypeptide that has been bound to a support. Apreferred support is beads or a plate. Preferably, the primary antibodyis a primary antibody that is labeled with a radioisotope or an enzyme.In another preferred embodiment, the contact between a TAK1 polypeptide,a TAB1 polypeptide and a test sample is carried out in a homogeneoussystem.

[0023] The present invention also provides (10) a method for screeningsubstances that inhibit binding between a TAK1 polypeptide and a TAB1polypeptide, which method comprises contacting the TAK1 polypeptide tothe TAB1 polypeptide and a test sample, and then detecting ordetermining the TAB1 polypeptide that is bound to the TAK1 polypeptideby a primary antibody against the TAB1 polypeptide. Preferably, the TAK1polypeptide is a TAK1 polypeptide that has been bound to a support. Apreferred support is beads or a plate. Preferably, the primary antibodyis a primary antibody that is labeled with a radioisotope, an enzyme ora fluorescent substance. In another preferred embodiment, the contactbetween a TAK1 polypeptide, a TAB1 polypeptide and a test sample iscarried out in a homogeneous system.

[0024] The present invention also provides (11) a screening methoddescribed in the above (9) and (10), which method comprises using a TAB1polypeptide having an amino acid sequence comprising Met at amino acidposition 1 to Pro at amino acid position 504 of the amino acid sequenceas set forth in SEQ ID NO: 2 or having an amino acid sequence modifiedby the substitution, deletion and/or addition of one or a plurality ofamino acid residues of the amino acid sequence as set forth in SEQ IDNO: 2 and maintaining the biological activity of the TAB1 polypeptide.

[0025] a TAK1 polypeptide having an amino acid sequence comprising Metat amino acid position 1 to Ser at amino acid position 579 of the aminoacid sequence as set forth in SEQ ID NO: 4 or having an amino acidsequence modified by the substitution, deletion and/or addition of oneor a plurality of amino acid residues of the amino acid sequence as setforth in SEQ ID NO: 4 and maintaining the biological activity of theTAK1 polypeptide.

[0026] The present invention also provides (12) a screening methoddescribed in the above (9) to (11), which method comprises using a TAK1polypeptide fused to another peptide or polypeptide and/or a TAB1polypeptide fused to another peptide or polypeptide.

[0027] The present invention also provides (13) a method for screeningsubstances that inhibit binding between a TAK1 polypeptide and a TAB1polypeptide, which method comprises contacting the TAB1 polypeptide orthe TAB1 polypeptide fused to another peptide or polypeptide to the TAK1polypeptide fused to another peptide or polypeptide and a test sample,and then detecting or determining the TAK1 polypeptide fused to anotherpeptide or polypeptide said TAK1 polypeptide being bound to the TAB1polypeptide or the TAB1 polypeptide fused to another peptide orpolypeptide by a primary antibody against the other peptide orpolypeptide. Preferably, the TAB1 polypeptide or the TAB1 polypeptidefused to another peptide or polypeptide is a TAB1 polypeptide or a TAB1polypeptide fused to another peptide or polypeptide, that has been boundto a support. A preferred support is beads or a plate. Preferably, theprimary antibody is a primary antibody that is labeled with aradioisotope or an enzyme. In another preferred embodiment, the contactbetween a TAK1 polypeptide, a TAB1 polypeptide and a test sample iscarried out in a homogeneous system.

[0028] The present invention also provides (14) a method for screeningsubstances that inhibit binding between a TAK1 polypeptide and a TAB1polypeptide, which method comprises contacting the TAK1 polypeptide orthe TAK1 polypeptide fused to another peptide or polypeptide to the TAB1polypeptide fused to another peptide or polypeptide and a test sample,and then detecting or determining the TAB1 polypeptide fused to anotherpeptide or polypeptide said TAB1 polypeptide being bound to the TAK1polypeptide or the TAK1 polypeptide fused to another peptide orpolypeptide, by a primary antibody against the other peptide orpolypeptide. Preferably, the TAK1 polypeptide or the TAK1 polypeptidefused to another peptide or polypeptide is a TAK1 polypeptide or a TAK1polypeptide fused to another peptide or polypeptide, that has been boundto a support. A preferred support is beads or a plate. Preferably, theprimary antibody is a primary antibody that is labeled with aradioisotope, an enzyme a fluorescent substance. In another preferredembodiment, the contact between a TAK1 polypeptide, a TAB1 polypeptideand a test sample is carried out in a homogeneous system.

[0029] The present invention also provides (15) a screening methoddescribed in the above (13) and (14), which method comprises using aTAB1 polypeptide having an amino acid sequence comprising Met at aminoacid position 1 to Pro at amino acid position 504 of the amino acidsequence as set forth in SEQ ID NO: 2 or having an amino acid sequencemodified by the substitution, deletion and/or addition of one or aplurality of amino acid residues of the amino acid sequence as set forthin SEQ ID NO: 2 and maintaining the biological activity of the TAB1polypeptide; and/or

[0030] a TAK1 polypeptide having an amino acid sequence comprising Metat amino acid position 1 to Ser at amino acid position 579 of the aminoacid sequence as set forth in SEQ ID NO: 4 or having an amino acidsequence modified by the substitution, deletion and/or addition of oneor a plurality of amino acid residues of the amino acid sequence as setforth in SEQ ID NO: 4 and maintaining the biological activity of theTAK1 polypeptide.

[0031] The present invention also provides (16) a method for screeningsubstances that inhibit binding between a TAK1 polypeptide and a TAB1polypeptide, which method comprises contacting the TAB1 polypeptide tothe TAK1 polypeptide and a test sample, and then detecting ordetermining the TAK1 polypeptide that is bound to the TAB1 polypeptideby a primary antibody against the TAK1 polypeptide and a secondaryantibody against the primary antibody. Preferably, the TAB1 polypeptideis a TAB1 polypeptide that has been bound to a support. A preferredsupport is beads or a plate. Preferably, the secondary antibody is asecondary antibody that is labeled with a radioisotope, an enzyme or afluorescent substance. In another preferred embodiment, the contactbetween a TAK1 polypeptide, a TAB1 polypeptide and a test sample iscarried out in a homogeneous system.

[0032] The present invention also provides (17) a method for screeningsubstances that inhibit binding between a TAK1 polypeptide and a TAB1polypeptide, which method comprises contacting the TAK1 polypeptide tothe TAB1 polypeptide and a test sample, and then detecting ordetermining the TAB1 polypeptide that is bound to the TAK1 polypeptideby a primary antibody against the TAB1 polypeptide and a secondaryantibody against the primary antibody. Preferably, the TAK1 polypeptideis a TAK1 polypeptide that has been bound to a support. A preferredsupport is beads or a plate. Preferably, the secondary antibody is asecondary antibody that is labeled with a radioisotope, an enzyme or afluorescent substance. In another preferred embodiment, the contactbetween a TAK1 polypeptide, a TAB1 polypeptide and a test sample iscarried out in a homogeneous system.

[0033] The present invention also provides (18) a screening methoddescribed in the above (16) and (17), which method comprises using aTAB1 polypeptide having an amino acid sequence comprising Met at aminoacid position 1 to Pro at amino acid position 504 of the amino acidsequence as set forth in SEQ ID NO: 2 or having an amino acid sequencemodified by the substitution, deletion and/or addition of one or aplurality of amino acid residues of the amino acid sequence as set forthin SEQ ID NO: 2 and maintaining the biological activity of the TAB1polypeptide; and/or

[0034] a TAK1 polypeptide having an amino acid sequence comprising Metat amino acid position 1 to Ser at amino acid position 579 of the aminoacid sequence as set forth in SEQ ID NO: 4 or having an amino acidsequence modified by the substitution, deletion and/or addition of oneor a plurality of amino acid residues of the amino acid sequence as setforth in SEQ ID NO: 4 and maintaining the biological activity of theTAK1 polypeptide.

[0035] The present invention also provides (19) a screening methoddescribed in the above (16) to (18), which method comprises using a TAK1polypeptide fused to another peptide or polypeptide and/or a TAB1polypeptide fused to another peptide or polypeptide.

[0036] The present invention also provides (20) a method for screeningsubstances that inhibit binding between a TAK1 polypeptide and a TAB1polypeptide, which method comprises contacting the TAB1 polypeptide orthe TAB1 polypeptide fused to another peptide or polypeptide to the TAK1polypeptide fused to another peptide or polypeptide and a test sample,and then detecting or determining the TAK1 polypeptide fused to anotherpeptide or polypeptide said TAK1 polypeptide being bound to the TAB1polypeptide or the TAB1 polypeptide fused to another peptide orpolypeptide, by a primary antibody against the other peptide orpolypeptide and a secondary antibody against the primary antibody.Preferably, the TAB1 polypeptide or the TAB1 polypeptide fused toanother peptide or polypeptide is a TAB1 polypeptide or the TAB1polypeptide fused to another peptide or polypeptide, that has been boundto a support. A preferred support is beads or a plate. Preferably, thesecondary antibody is a primary antibody that is labeled with aradioisotope, an enzyme or a fluorescent substance. In another preferredembodiment, the contact between a TAK1 polypeptide, a TAB1 polypeptideand a test sample is carried out in a homogeneous system.

[0037] The present invention also provides (21) a method for screeningsubstances that inhibit binding between a TAK1 polypeptide and a TAB1polypeptide, which method comprises contacting the TAK1 polypeptide orthe TAK1 polypeptide fused to another peptide or polypeptide to the TAB1polypeptide fused to another peptide or polypeptide and a test sample,and then detecting or determining the TAB1 polypeptide fused to anotherpeptide or polypeptide said TAB1 polypeptide being bound to the TAK1polypeptide or the TAK1 polypeptide fused to another peptide orpolypeptide, by a primary antibody against the other peptide orpolypeptide and a secondary antibody against the primary antibody.Preferably, the TAK1 polypeptide or the TAK1 polypeptide fused toanother peptide or polypeptide is a TAK1 polypeptide or the TAK1polypeptide fused to another peptide or polypeptide, that has been boundto a support. A preferred support is beads or a plate. Preferably, thesecondary antibody is a secondary antibody that is labeled with aradioisotope, an enzyme a fluorescent substance. In another preferredembodiment, the contact between a TAK1 polypeptide, a TAB1 polypeptideand a test sample is carried out in a homogeneous system.

[0038] The present invention also provides (22) a screening methoddescribed in the above (20) and (21), which method comprises using aTAB1 polypeptide having an amino acid sequence comprising Met at aminoacid position 1 to Pro at amino acid position 504 of the amino acidsequence as set forth in SEQ ID NO: 2 or having an amino acid sequencemodified by the substitution, deletion and/or addition of one or aplurality of amino acid residues of the amino acid sequence as set forthin SEQ ID NO: 2 and maintaining the biological activity of the TAB1polypeptide; and/or

[0039] a TAK1 polypeptide having an amino acid sequence comprising Metat amino acid position 1 to Ser at amino acid position 579 of the aminoacid sequence as set forth in SEQ ID NO: 4 or having an amino acidsequence modified by the substitution, deletion and/or addition of oneor a plurality of amino acid residues of the amino acid sequence as setforth in SEQ ID NO: 4 and maintaining the biological activity of theTAK1 polypeptide.

[0040] The present invention also provides a kit for conducting thescreening method as set forth in any of the above (1) to (22).

[0041] The present invention also provides a substance that isobtainable by the screening method as set forth in any of the above (1)to (22).

[0042] The present invention also provides a substance that inhibitsbinding between a TAK1 polypeptide and a TAB1 polypeptide obtainable bythe screening method as set forth in any of the above (1) to (22).

[0043] The present invention also provides an inhibitor of signaltransduction of TGF-β, said inhibitor comprising a substance thatinhibits binding between a TAK1 polypeptide and a TAB1 polypeptide,obtainable by the screening method as set forth in any of the above (1)to (22).

[0044] The present invention also provides an activator of signaltransduction of TGF-β, said activator comprising a substance thatinhibits binding between a TAK1 polypeptide and a TAB1 polypeptide,obtainable by the screening method as set forth in any of the above (1)to (22).

[0045] The present invention also provides a suppressor of theenhancement of extracellular matrix protein production, said suppressorcomprising, as an active ingredient, a substance that inhibits bindingbetween a TAK1 polypeptide and a TAB1 polypeptide, obtainable by thescreening method as set forth in any of the above (1) to (22).

[0046] The present invention also provides an activator of theenhancement of extracellular matrix protein production, said activatorcomprising, as an active ingredient, a substance that inhibits bindingbetween a TAK1 polypeptide and a TAB1 polypeptide, obtainable by thescreening method as set forth in any of the above (1) to (22).

[0047] The present invention also provides a suppressor of theinhibition of cellular growth, said suppressor comprising, as an activeingredient, a substance that inhibits binding between a TAK1 polypeptideand a TAB1 polypeptide, obtainable by the screening method as set forthin any of the above (1) to (22).

[0048] The present invention also provides an activator of theinhibition of cellular growth, said activator comprising, as an activeingredient, a substance that inhibits binding between a TAK1 polypeptideand a TAB1 polypeptide, obtainable by the screening method as set forthin any of the above (1) to (22).

[0049] The present invention also provides a suppressor of monocytemigration, said suppressor comprising, as an active ingredient, asubstance that inhibits binding between a TAK1 polypeptide and a TAB1polypeptide, obtainable by the screening method as set forth in any ofthe above (1) to (22).

[0050] The present invention also provides an activator of monocytemigration, said activator comprising, as an active ingredient, asubstance that inhibits binding between a TAK1 polypeptide and a TAB1polypeptide, obtainable by the screening method as set forth in any ofthe above (1) to (22).

[0051] The present invention also provides a suppressor of the inductionof a biologically active substance, said suppressor comprising, as anactive ingredient, a substance that inhibits binding between a TAK1polypeptide and a TAB1 polypeptide, obtainable by the screening methodas set forth in any of the above (1) to (22).

[0052] The present invention also provides an activator of abiologically active substance, said activator comprising, as an activeingredient, a substance that inhibits binding between a TAK1 polypeptideand a TAB1 polypeptide, obtainable by the screening method as set forthin any of the above (1) to (22).

[0053] The present invention also provides a suppressor of animmunosuppressive action, said suppressor comprising, as an activeingredient, a substance that inhibits binding between a TAK1 polypeptideand a TAB1 polypeptide, obtainable by the screening method as set forthin any of the above (1) to (22).

[0054] The present invention also provides an activator of animmunosuppressive action, said activator comprising, as an activeingredient, a substance that inhibits binding between a TAK1 polypeptideand a TAB1 polypeptide, obtainable by the screening method as set forthin any of the above (1) to (22).

[0055] The present invention also provides a suppressor of thedeposition of amyloid β protein, said suppressor comprising, as anactive ingredient, a substance that inhibits binding between a TAK1polypeptide and a TAB1 polypeptide, obtainable by the screening methodas set forth in any of the above (1) to (22).

[0056] The present invention also provides an activator of thedeposition of amyloid β protein, said activator comprising, as an activeingredient, a substance that inhibits binding between a TAK1 polypeptideand a TAB1 polypeptide, obtainable by the screening method as set forthin any of the above (1) to (22).

BRIEF EXPLANATION OF DRAWINGS

[0057]FIG. 1 is a diagram showing the construction of human TAB1-FLAGand human TAK1-6xHis.

[0058]FIG. 2 is a graph showing binding between human TAK1-FLAG andhuman MBP-TAB1C-FLAG.

[0059]FIG. 3 is a graph showing binding between human TAB1-FLAG andhuman TAK1-6xHis.

[0060]FIG. 4 is a graph showing the activity of inhibition of bindingbetween human TAK1-6xHis and human MBP-TAB1C-FLAG, determined usingTAB1-FLAG as an inhibiting substance.

[0061]FIG. 5A is a graph showing the amount of fibronectin determined inthe culture supernatant of the HT/NEO cells, the HT/DN2 cells and theHT/DN14 cells with and without the addition of TGF-β1. The valuesrepresent the mean +/− S.D. of the amount of fibronectin in the culturesupernatant prepared from three different wells. FIG. 5B is a graphshowing the amount of fibronectin determined in the matrix extract ofthe HT/NEO cells, the HT/DN2 cells and the HT/DN14 cells with andwithout the addition of TGF-β1. The values represent the mean +/− S.D.of the amount of fibronectin in the matrix extract prepared from threedifferent wells.

[0062]FIG. 6A is a graph showing the amount of fibronectin determined inthe culture supernatant of the MES/NEO cells, the MES/DN3 cells and theMES/DN6 cells with and without the addition of TGF-β1. The valuesrepresent the mean +/− S.D. of the amount of fibronectin in the culturesupernatant prepared from three different wells. FIG. 6B is a graphshowing the amount of fibronectin determined in the matrix extract ofthe MES/NEO cells, the MES/DN3 cells and the MES/DN6 cells with andwithout the addition of TGF-β1. The values represent the mean +/− S.D.of the amount of fibronectin in the matrix extract prepared from threedifferent wells.

[0063]FIG. 7 is a graph showing the amount of type I collagen determinedin the culture supernatant of the MES/NEO cells, the MES/DN3 cells andthe MES/DN6 cells with and without the addition of TGF-β1. The valuesrepresent the mean +/−S.D. of the amount of type I collagen in theculture supernatant prepared from three different wells.

[0064]FIG. 8 is a graph showing the amount of type IV collagendetermined in the culture supernatant of the MES/NEO cells, the MES/DN3cells and the MES/DN6 cells with and without the addition of TGF-β1. Thevalues represent the mean +/− S.D. of the amount of type IV collagen inthe culture supernatant prepared from three different wells.

[0065]FIG. 9 is a graph showing the result of a two-hybrid assay usingthe CHO cells. The values represent the mean +/− S.D. of the luciferaseactivity in the culture supernatant prepared from three different wells.

[0066]FIG. 10 is a graph showing the amount of PAI-1 in the culturesupernatant when TGF-β1 was added to the Mv1Lu cells. The valuesrepresent the mean +/− S.D. of the amount of PAI-1 in the culturesupernatant prepared from three different wells.

[0067]FIG. 11 is the activity in Miller Units of β-galactosidase of ayeast L40 that was transformed with an amino terminal-truncated TAB1mutants (TAB1C45^(—)TAB1C20) and the yeast 2-hybrid expression plasmidof TAK1. The measurement was conducted three times and the result isexpressed in the mean +/− S.D. The values represent a ratio based on theβ-galactosidase activity of the yeast L40 that was transformed withTAB1C68 and the yeast 2-hybrid expression plasmid of TAK1.

[0068]FIG. 12 is the activity in Miller Units of β-galactosidase of ayeast L40 that was transformed with a carboxy terminal-truncated TAB1mutants (TAB1C45 Δ14^(—)TAB1C45Δ25) and a yeast 2-hybrid expressionplasmid of TAK1. The measurement was conducted three times and theresult is expressed in the mean +/− S.D. The values represent a ratio tothe β-galactosidase activity of the yeast L40 that was transformed withTAB1C68 and the yeast 2-hybrid expression plasmid of TAK1.

[0069]FIG. 13A is the result of Western analysis of TAK1 and FLAG-TAB1contained in the immunoprecipitate obtained using anti-TAK1 antibody inthe presence or absence of each peptide. FIG. 13B is the result obtainedby quantifying the density of bands each obtained by Western analysisand then by correcting the amount of the co-precipitated FLAG-TAB1 withthe amount of TAK1. The values represent values relative to thatobtained in the absence of the peptide which was set as 1.

[0070]FIG. 14 shows the ability of the TAB1 deletion mutants (TAB1C68,TAB1C45, TAB1C40, TAB1C35, TAB1C30 and TAB1C25) to bind to and activateTAK1.

EMBODIMENT FOR CARRYING OUT THE INVENTION

[0071] The TAB1 polypeptide for use in the present invention may be anyTAB1 polypeptide, as long as it has an amino acid sequence comprisingMet at amino acid position 1 to Pro at amino acid position 504 of theamino acid sequence as set forth in SEQ ID NO: 2 and the biologicalactivity of the TAB1. It has been demonstrated that the biologicalactivity of the TAB1 polypeptide is the activity of binding to andactivating the TAK1 polypeptide.

[0072] More specifically, it has been demonstrated that the biologicalactivity of the TAB1 polypeptide is the activity of binding to a regioncontaining the catalytic domain of the TAK1 polypeptide having an aminoacid sequence comprising an amino acid Met at position 1 to an aminoacid Glu at position 303 of the TAK1 polypeptide and activating thekinase activity of the TAK1 polypeptide to the MAPKK. In the presentinvention, however, the TAB1 polypeptide is only required to have theactivity of binding to the TAK1 polypeptide and may be a TAB1polypeptide that has lost the activity of activating the TAK1polypeptide. Accordingly, the biological activity of the TAB1polypeptide as used herein may be the activity of binding to the TAK1polypeptide.

[0073] The TAB1 polypeptide for use in the present invention may be aTAB1 polypeptide that has the biological activity of the TAB1polypeptide and that has an amino acid sequence modified by thesubstitution, deletion and/or addition of one or a plurality of aminoacid residues of the amino acid sequence as set forth in SEQ ID NO: 2.More specifically, the TAB1 polypeptide for use in the present inventionmay have an amino acid sequence in which one or more than one,preferably one or not greater than 20, and more preferably one or notgreater than 10 amino acid residues are substituted in the amino acidsequence as set forth in SEQ ID NO: 2, as long as it has thebiologically activity of the TAB1 polypeptide.

[0074] Alternatively, the amino acid sequence as set forth in SEQ ID NO:2 may be modified by deletion of one or more than one, preferably one ornot greater than 436, and more preferably one or not greater than 10amino acid residues. The amino acid sequence as set forth in SEQ ID NO:2 may also be modified by addition of one or more than one, preferablyone or not greater than 30, and more preferably one or not greater than20 amino acid residues. The TAB1 polypeptide for use in the presentinvention may also be modified by simultaneous substitution, deletion,and/or addition of the above amino acids.

[0075] It has been elucidated that the TAB1 polypeptide exhibits itsbiological activity as long as it has an amino acid sequence comprisingamino acid Gln at position 437 to amino acid Pro at position 504 in SEQID NO: 2. Thus, the TAB1 polypeptide for use in the present inventionmay be a TAB1 polypeptide that has an amino acid sequence comprisingamino acid Gln at position 437 to amino acid Pro at position 504 in SEQID NO: 2, or has an amino acid sequence modified by the substitution,deletion and/or addition of one or a plurality of amino acid residues inthe amino acid sequence comprising amino acid Met at position 1 to aminoacid Asn at position 436.

[0076] The TAB1 polypeptide may be a TAB1 polypeptide that has an aminoacid sequence modified by the substitution, deletion and/or addition ofone or a plurality of amino acid residues in the amino acid sequencecomprising amino acid Gln at position 437 to amino acid Pro at position504 in SEQ ID NO: 2, as long as it has the biological activity of theTAB1 polypeptide.

[0077] As a TAB1 polypeptide that has an amino acid sequence modified bythe substitution, deletion and/or addition of one or a plurality ofamino acid residues in the amino acid sequence as set forth in SEQ IDNO: 2, there can be mentioned a TAB1 polypeptide in which amino acid Serat position 52 has been replaced with Arg and a TAB1 polypeptide thathas an amino acid sequence comprising amino acid Gln at position 437 toamino acid Pro at position 504.

[0078] It is already known that a polypeptide that has an amino acidsequence modified by the substitution, deletion and/or addition of oneor a plurality of amino acid residues of an amino acid sequence retainsits biological activity (Mark, D. F. et al., Proc. Natl. Acad. Sci.U.S.A. (1984) 81, 5662-5666; Zoller, M. J. & Smith, M. Nucleic AcidsResearch (1982) 10, 6487-6500; Wang, A. et al., Science 224, 1431-1433;Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci. U.S.A. (1982) 79,6409-6413).

[0079] The TAK1 polypeptide for use in the present invention may be anyTAK1 polypeptide al long as it has an amino acid sequence comprising Metat amino acid position 1 to Ser at amino acid position 579 in the aminoacid sequence as set forth in SEQ ID NO: 4 and the biological activityof TAK1. It has been demonstrated that the biological activity of theTAK1 polypeptide is the activity of binding to the TAB1 polypeptide andthe kinase activity to MAPKK at an activated state.

[0080] More specifically, it has been demonstrated that it is theactivity of activating the kinase activity of MAPKK by exhibiting thekinase activity at an activated state thereby phosphorylating MAPKK, forexample MKK3 (Moriguchi, T. et al., J. Biol. Chem. (1996) 271,13675-13679) and XMEK2/SEKI (Shibuya, H. et al., Science (1996) 272,1179-1182). In the present invention, however, the TAK1 polypeptide isonly required to have the activity of binding to the TAB1 polypeptideand may be a TAK1 polypeptide that has lost the kinase activity of theTAK1 polypeptide. Accordingly, the biological activity of the TAK1polypeptide as used herein may be the activity of binding to the TAB1polypeptide.

[0081] It has been elucidated that the TAK1 polypeptide exhibits itsbiological activity as long as it has an amino acid sequence comprisingamino acid Met at position 1 to amino acid Gln at position 303 in SEQ IDNO: 4. Thus, the TAK1 polypeptide for use in the present invention maybe a TAK1 polypeptide that has an amino acid sequence comprising aminoacid Met at position 1 to amino acid Gln at position 303 in SEQ ID NO:4, and an amino acid sequence modified by the substitution, deletionand/or addition of one or a plurality of amino acid residues in theamino acid sequence comprising amino acid Tyr at position 304 from aminoacid Tyr to amino acid Ser at position 579. The TAK1 polypeptide may bea TAK1 polypeptide that has an amino acid sequence modified by thesubstitution, deletion and/or addition of one or a plurality of aminoacid residues in the amino acid sequence comprising amino acid Met atposition 1 to amino acid Gln at position 303, as long as it has thebiological activity of the TAK1 polypeptide.

[0082] The TAK1 polypeptide is activated by the binding of the TAB1polypeptide to a region containing a catalytic domain of a TAK1polypeptide that has an amino acid sequence comprising amino acid Met atposition 1 to amino acid Glu at position 303 of the TAK1 polypeptide asset forth in SEQ ID NO: 4. In accordance with the present invention, ithas been disclosed that the TAK1 polypeptide binds to the TAB1polypeptide at the amino acid sequence comprising amino acid Val atposition 76 to amino acid Gln at position 303 of the TAK1 polypeptide asset forth in SEQ ID NO: 4. Although the TAK1 polypeptide that has anamino acid sequence comprising amino acid Val at position 76 to aminoacid Gln at position 303 of the TAK1 polypeptide as set forth in SEQ IDNO: 4 did not exhibit any kinase activity, it has the activity ofbinding to the TAB1 polypeptide, and therefore it can be used in thepresent invention.

[0083] Thus, it may be a TAK1 polypeptide that has an amino acidsequence comprising amino acid Val at position 76 to amino acid Gln atposition 303 in SEQ ID NO: 4 and an amino acid sequence modified by thesubstitution, deletion and/or addition of one or a plurality of aminoacid residues in the amino acid sequence comprising amino acid Met atposition 1 to amino acid Ile at position 75 and amino acid Tyr atposition 304.

[0084] The TAK1 polypeptide may be a TAK1 polypeptide that has an aminoacid sequence modified by the substitution, deletion and/or addition ofone or a plurality of amino acid residues in the amino acid sequencecomprising amino acid Val at position 76 to amino acid Gln at position303 in SEQ ID NO: 4, as long as it has the activity of binding to theTAB1 polypeptide. The biological activity of the TAK1 polypeptide mayalso be activated by deleting at least 21 amino acid residues at theamino group-side terminal (N-terminal) of the TAK1 polypeptide.

[0085] The TAK1 polypeptide for use in the present invention may be aTAK1 polypeptide that has the biological activity of TAK1 polypeptideand an amino acid sequence modified by the substitution, deletion and/oraddition of one or a plurality of amino acid residues in the amino acidsequence as set forth in SEQ ID NO: 4. More specifically, the TAK1polypeptide for use in the present invention may have amino acids thatare substituted with one or more than one, preferably one or not greaterthan 20, and more preferably one or not greater than 10 amino acidresidues in the amino acid sequence as set forth in SEQ ID NO: 4, aslong as it has the biologically activity of the TAK1 polypeptide.

[0086] Alternatively, the amino acid sequence as set forth in SEQ ID NO:4 may have amino acids in which one or more than one, preferably one ornot greater than 276, and more preferably one or not greater than 10amino acid residues are deleted. Or, the amino acid sequence as setforth in SEQ ID NO: 4 may have amino acids in which one or more thanone, preferably one or not greater than 30, and more preferably one ornot greater than 20 amino acid residues are added.

[0087] As a TAK1 polypeptide that has an amino acid sequence modified bythe substitution, deletion and/or addition of one or a plurality ofamino acid residues in the amino acid sequence as set forth in SEQ IDNO: 4, there can be mentioned a TAK1 polypeptide of a mouse origin inwhich amino acid Gly at position 16 is replaced with Ser, amino acid Hisat position 372 is replaced with Arg, amino acid Ala at position 400 isreplaced with Val, amino acid Thr at position 403 is replaced with Ala,and amino acid Thr at position 449 is replaced with Ala.

[0088] It is already known that a polypeptide that has an amino acidsequence modified by the substitution, deletion and/or addition of oneor a plurality of amino acid residues of an amino acid sequence retainsits biological activity (Mark, D. F. et al., Proc. Natl. Acad. Sci.U.S.A. (1984) 81, 5662-5666; Zoller, M. J. & Smith, M. Nucleic AcidsResearch (1982) 10, 6487-6500; Wang, A. et al., Science 224, 1431-1433;Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci. U.S.A. (1982) 79,6409-6413).

[0089] The polypeptides for use in the present invention differ in theamino acid sequence, molecular weight, isoelectric point, the presenceor absence of an added sugar chain, the position of an added sugarchain, the structure of a sugar chain, the state of phosphorylation,and/or the presence or absence of a disulfide bond depending on thespecies from which they are derived, the host that produces them, and/orthe method of purification. However, polypeptides having any structuremay be used as long as they can be suitably used in the presentinvention. Preferably, the species from which the polypeptide is derivedis human.

[0090] As DNA encoding the TAB1 polypeptide for use in the presentinvention, there may be mentioned a nucleotide sequence comprising baseA at nucleotide position 30 to nucleotide G at position 1541 of thenucleotide sequence as set forth in SEQ ID NO: 1. Furthermore, DNAencoding the TAB1 polypeptide for use in the present invention can be ofany origin as long as it has the base sequence as set forth in SEQ IDNO: 1. Such DNA includes, for example, genomic DNA, cDNA, and syntheticDNA. They may be DNA obtained from a cDNA library and a genomic libraryobtained from various cells, tissues, or organs, or from species otherthan humans, and they may be a commercially available DNA library.Vectors for use in such libraries may be plasmids, bacteriophages, YACvectors, and the like.

[0091] DNA encoding the TAB1 polypeptide for use in the presentinvention may be DNA that hybridizes to the nucleotide sequence as setforth in SEQ ID NO: 1 and encodes a polypeptide having the biologicalactivity of TAB1 . As a condition under which DNA encoding the TAB1polypeptide hybridizes, there may be mentioned a stringent condition.

[0092] Such conditions include, for example, a low stringent condition.By way of example, a low stringent condition is a washing conditionprovided at room temperature in 2 × SSC and 0.1% sodium dodecyl sulfate.More preferably, there may be mentioned a high stringent condition. Byway of example, a high stringent condition is a washing conditionprovided at 60° C. in 0.1 × SSC and 0.1% sodium dodecyl sulfate. It isalready known that a polypeptide encoded by a DNA that hybridizes undera suitable condition to a base sequence encoding a polypeptide has thesame biological activity as the polypeptide.

[0093]E. coli that has the plasmid TAB1-f-4 containing DNA encoding thehuman TAB1 polypeptide having an amino acid sequence comprising aminoacid Met at amino acid position 1 to amino acid Pro at amino acidposition 504 of the amino acid sequence as set forth in SEQ ID NO: 2 wasdesignated as Escherichia coli DH5α (TABI-f-4) and has beeninternationally deposited under the provisions of the Budapest Treaty onJul. 19, 1996, with the National Institute of Bioscience and HumanTechnology, Agency of Industrial Science and Technology, of 1-3, Higashi1-chome, Tsukuba-shi, Ibaraki, Japan, as the accession number FERMBP-5599.

[0094]E. coli that has the plasmid pBS-TAB1 containing DNA encoding theabove human TAB1 polypeptide that comprises amino acid Met at amino-acidposition 1 to amino acid Pro at amino acid position 504 of the aminoacid sequence as set forth in SEQ ID NO: 2 and in which amino acid Serat position 52 has been replaced with Arg was designated as Escherichiacoli HB101 (pBS-TAB1) and has been internationally deposited under theprovisions of the Budapest Treaty on Apr. 19, 1996, with the NationalInstitute of Bioscience and Human Technology, Agency of IndustrialScience and Technology, of 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki,Japan, as the accession number FERM BP-5508.

[0095] As DNA encoding the TAK1 polypeptide for use in the presentinvention, there may be mentioned a nucleotide sequence comprisingnucleotide A at nucleotide position 183 to nucleotide G at position 1919of the nucleotide sequence as set forth in SEQ ID NO: 2. Furthermore,DNA encoding the TAK1 polypeptide for use in the present invention canbe of any origin as long as it has the nucleotide sequence as set forthin SEQ ID NO: 3. Such a DNA includes, for example, genomic DNA, cDNA,and synthetic DNA.

[0096] They may be DNA obtained from a cDNA library and a genomiclibrary obtained from various cells, tissues, or organs, or from speciesother than human, and they may be a commercially available DNA library.Vectors for use in such libraries may be plasmids, bacteriophages, YACvectors, and the like.

[0097] DNA encoding the TAK1 polypeptide for use in the presentinvention may be DNA that hybridizes to the nucleotide sequence as setforth in SEQ ID NO: 3 and encodes a polypeptide having the biologicalactivity of TAK1. As a condition under which the DNA encoding the TAB1polypeptide hybridizes, there may be mentioned a stringent condition.

[0098] Such conditions include, for example, a low stringent condition.By way of example, a low stringent condition is a washing conditionprovided at room temperature in 2 × SSC and 0.1% sodium dodecyl sulfate.More preferably, there may be mentioned a high stringent condition. Byway of example, a high stringent condition is a washing conditionprovided at 60° C. in 0.1 × SSC and 0.1% sodium dodecyl sulfate. It isalready known that a polypeptide encoded by a DNA that hybridizes undera suitable condition to a nucleotide sequence encoding a polypeptide hasthe same biological activity as the polypeptide.

[0099]E. coli that has the plasmid pEF-TAK1 containing DNA encoding theabove mouse TAK1 polypeptide was designated as Escherichia coliMC1061/P3 (pEF-TAK1) and has been internationally deposited under theprovisions of the Budapest Treaty on Sep. 28, 1995, with the NationalInstitute of Bioscience and Human Technology, Agency of IndustrialScience and Technology, of 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki,Japan, as the accession number FERM BP-5246.

[0100]E. coli that has the plasmid pEF-TAK1DN containing DNA encodingthe mouse TAK1 polypeptide that has a deletion of 21 amino acids at theN-terminal was designated as Escherichia coli MC1061/P3 (pEF-TAK1DN) andhas been internationally deposited under the provisions of the BudapestTreaty on Sep. 28, 1995, with the National Institute of Bioscience andHuman Technology, Agency of Industrial Science and Technology, of 1-3,Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan, as the accession numberFERM BP-5245.

[0101]E. coli that has the plasmid phTAK1 containing DNA encoding thehuman TAK1 polypeptide that has an amino acid sequence comprising aminoacid Met at amino acid position 1 to amino acid Ser at amino acidposition 579 of the amino acid sequence as set forth in SEQ ID NO: 4 andwas designated as Escherichia coli JM109 (phTAK1) and has beeninternationally deposited under the provisions of the Budapest Treaty onJul. 19, 1996, with the National Institute of Bioscience and HumanTechnology, Agency of Industrial Science and Technology, of 1-3, Higashi1-chome, Tsukuba-shi, Ibaraki, Japan, as the accession number FERMBP-5598.

[0102] Polypeptides for use in the present invention may be the abovepolypeptides that are fused to another peptide or polypeptide. Suchfusion polypeptides may be produced by a known method. Another peptideor polypeptide subjected to fusion with the polypeptide may be anypeptide or polypeptide as long as it can be advantageously used in thepresent invention. As such peptides, for example, known peptides may beused including FLAG (Hopp, T. P. et al., BioTechnology (1988) 6,1204-1210), 6xHis comprising 6 His (histidine) residues, 10xHis,influenza hemaglutinin (HA), fragments of human c-myc, fragments ofVSV-GP, fragments of p18HIV, T7-tag, HSV-tag, E-tag, fragments of SV40Tantigen, lck tag, fragments of a-tubulin, B-tag, fragments of Protein C,and the like.

[0103] As polypeptides, there may be mentioned, for example, GST(glutathione S-transferase), HA, the constant regions of immunoglobulin,β-galactosidase, MBP (maltose-binding protein), and the like. They maybe commercially available polypeptides.

[0104] DNA encoding the polypeptide for use in the present invention maybe generated by constructing the above-mentioned DNA using commerciallyavailable kits or by known methods. There may be mentioned, for example,digestion with a restriction enzyme, addition of a linker, insertion ofan initiation codon (ATG) and/or a stop codon (ATT, TGA or TAG), and thelike.

[0105] Expression vectors for use in the present invention may be anyexpression vectors as long as they can be suitably used in the presentinvention. As expression vectors, there may be mentioned expressionvectors. derived from a mammal such as pEF and pCDM8, expression vectorsderived from an insect such as pBacPAK8, expression vectors derived froma plant such as pMH1 and pMH2, expression vectors derived from an animalvirus such as pHSV and pMV, expression vectors derived from a yeast suchas pNV11, expression vectors derived from Bacillus subtilis such aspPL608 and pKTH50 and expression vectors derived from Escherichia colisuch as pGEX, pGEMEX and pMALp2.

[0106] Expression vectors of polypeptides for use in the presentinvention may be produced by linking DNA encoding the TAB1 polypeptideor the TAK1 polypeptide downstream to the promoter. Aspromoters/enhancers, promoters/enhancers derived from a mammal such asthe EF1-α promoter/enhancer and γ-actin promoter/enhancer,promoters/enhancers derived from an insect such as polyhedrin viruspromoter/enhancer, promoters/enhancers derived from a plant such astabacco mosaic virus promoter/enhancer, promoters/enhancers derived froma plant such as SV40 promoter/enhancer and human CMV promoter/enhancer,promoters/enhancers derived from yeast such as alcohol dehydrogenasepromoter/enhancer, promoters/enhancers derived from Escherichia colisuch as Lac promoter/enhancer, Trp promoter/enhancer and TacPromoter/enhancer.

[0107] For the expression of polypeptide for use in the presentinvention, a signal sequence suitable for the host to be used in theexpression may be added. As the signal sequence, there may be mentioneda signal sequence for secretary proteins. As a signal sequence forsecretary proteins, there may be mentioned a signal sequence forsecretary proteins derived from a mammal such as a signal sequence forimmunoglobulins. As a signal sequence for secretary proteins, there maybe mentioned a signal sequence for secretary proteins derived from E.coli such as periplasm secretary signal sequence such as OmpA and thelike.

[0108] An expression vector produced as mentioned above can beintroduced into a host by a known method. Methods for introduction intothe host includes, for example, electropolation, the calcium phosphatemethod, and the liposome method.

[0109] Polypeptides for use in the present invention can be obtained asrecombinant polypeptides produced using gene recombinant technology asdescribed above. For example, recombinant polypeptides may be producedby cloning the base sequence of a gene described herein from a cell,tissue, or an organ that expresses the polypeptide and integrating thegene into a suitable vector, which is introduced into a host to allowthe host to produce said polypeptide. The recombinant polypeptides canbe used in the present invention.

[0110] Specifically, mRNA encoding the gene can be isolated from thecell, tissue, or organ that expresses polypeptides to be used in thepresent invention. The isolation of mRNA is conducted by preparing totalRNA using a known method such as the guanidine ultracentrifugationmethod (Chirgwin, J. M. et al., Biochemistry (1979) 18, 5294-5299), theAGPC method (Chomzynski, P. and Sacci, N., Anal. Biochem. (1987) 162,156-159), and then purifying mRNA from the total RNA using the mRNAPurification Kit (Pharmacia) and the like. Alternatively, mRNA can beprepared directly using the QuickPrep mRNA Purification kit (Pharmacia).

[0111] The mRNA obtained is used to synthesize the cDNA of the geneusing a reverse transcriptase. The synthesis of cDNA can be effectedusing the AMV Reverse Transcriptase First-strand cDNA Synthesis Kit(Seikagaku Kogyo), and the like. Alternatively, for the synthesis andamplification of cDNA, the Marathon cDNA Amplification kit (manufacturedby CLONTECH) and the 5′-RACE method (Frohman, M. A. et al., Proc. Natl.Acad. Sci. U.S.A. (1988) 85, 8998-9002; Belyavsky, A. et al., NucleicAcids Res. (1989) 17, 2919-2932) that employs the polymerase chainreaction (PCR) may be used.

[0112] A DNA fragment of interest may be prepared from the PCR productthus obtained and ligated to a vector DNA. Furthermore, a recombinantvector is constructed from this and is then introduced into E. coli forselection of colonies to prepare the desired recombinant vector. Thenucleotide sequence of the desired DNA may be confirmed by a knownmethod such as the dideoxy nucleotide chain termination method. Once thedesired DNA has been obtained, it may be integrated into an expressionvector.

[0113] More specifically, the DNA constructed as described above may beexpressed to obtain polypeptides. When mammalian cells are used,expression may be accomplished using a vector containing a commonly useduseful promoter/enhancer, the gene to be expressed, and DNA in which thepoly A signal has been operably linked at 3′ downstream thereof or avector containing said DNA. Examples of the promoter/enhancer includethe human cytomegalovirus immediate early promoter/enhancer.

[0114] Additionally, as the promoter/enhancer which can be used forexpression thereof, there are viral, promoters/enhancers such asretrovirus, polyoma virus, adenovirus, and simian virus 40 (SV40), andpromoters/enhancers derived from mammalian cells such as humanelongation factor 1α (HEF1α).

[0115] For example, expression may be readily accomplished by the methodof Mulligan et al. (Nature (1979) 277, 108) when the SV40promoter/enhancer is used, or by the method of Mizushima et al. (NucleicAcids Res. (1990) 18, 5322) when the HEF1α promoter/enhancer is used.

[0116] In the case of E. coli, expression may be conducted by operablylinking a commonly used useful promoter, a signal sequence forpolypeptide secretion, and the gene to be expressed, followed byexpression thereof. As the promoter, for example, there can be mentionedthe lacz promoter and the arab promoter. The method of Ward et al.(Nature (1098) 341, 544-546; FASEB J. (1992) 6, 2422-2427) may be usedwhen the lacz promoter is used, and the method of Better et al. (Science(1988) 240, 1041-1043) may be used when the araB promoter is used.

[0117] As a signal sequence for polypeptide secretion, when produced inthe periplasm of E. coli, the pelB signal sequence (Lei, S. P. et al.,J. Bacteriol. (1987) 169, 4379) can be used.

[0118] As a origin of replication, there can be used those derived fromSV40, polyoma virus, adenovirus, bovine papilloma virus (BPV) and thelike. Furthermore, for the amplification of gene copy number in the hostcell system, expression vectors can include as selectable markers theaminoglycoside transferase (APH) gene, the thymidine kinase (TK) gene,E. coli xanthine guaninephosphoribosyl transferase (Ecogpt) gene, thedihydrofolate reductase (dhfr) gene and the like.

[0119] For the production of polypeptides for use in the presentinvention, any production system can be used. The production system ofpolypeptide preparation comprises the in vitro or the in vivo productionsystem. As the in vitro production system, there can be mentioned aproduction system which employs eukaryotic cells and the productionsystem which employs prokaryotic cells.

[0120] When the eukaryotic cells are used, there are the productionsystems which employ the animal cells, the plant cells, and the fungalcells. Known animal cells include (1) mammalian cells such as CHO cells(J. Exp. Med. (1995) 108, 945), COS cells, myeloma cells, baby hamsterkidney (BHK) cells, HeLa cells, and Vero cells, (2) amphibian cells suchas xenopus oocytes (Valle, et al., Nature (1981) 291, 358-340), or (3)insect cells such as sf9, sf21, and Tn5. As the CHO cells, dhfr-CHO(Proc. Natl. Acad. Sci. U.S.A. (1968) 77, 4216-4220), a CHO cell that isdeficient in the DHFR gene, and CHO K-1 (Proc. Natl. Acad. Sci. U.S.A.(1968) 60, 1275) can be preferably used.

[0121] Known plant cells include, for example, those derived fromNicotiana tabacum, which is subjected to callus culture. Known fungalcells include yeasts such as the genus Saccharomyces, more specificallySaccharomyces cereviceae, or filamentous fungi such as the genusAspergillus, more specifically Aspergillus nicer.

[0122] When the prokaryotic cells are used, there are the productionsystems which employ bacterial cells. Known bacterial cells includeEscherichia coli (E. coli), and Bacillus subtilis.

[0123] By transforming these cells with the desired DNA and culturingthe transformed cells in vitro, polypeptides can be obtained. Culturingis conducted in the known methods. For example, as the culture liquid,DMEM, MEM, RPMI1640, and IMDM can be used, and serum supplements such asfetal calf serum (FCS) may be used in combination, or serum-free mediumcan be used. pH during the culture is preferably about 6 to 8. Cultureis usually carried out at 30 to 40° C. for about 15 to 200 hours with,as desired, medium changes, aeration, and agitation.

[0124] As in vivo production systems, there can be mentioned those whichemploy animals and those which employ plants. DNA of interest isintroduced into these animals or plants, and the polypeptides areproduced in such animals or plants, and recovered.

[0125] When animals are used, there are the production systems whichemploy mammals and insects.

[0126] As mammals, goat, pigs, sheep, mice, and cattle can be used(Vicki Glaser, SPECTRUM Biotechnology Applications, 1993). When mammalsare used, transgenic animals can be used.

[0127] For example, a DNA of interest is inserted into the middle of thegene encoding protein which is inherently produced in the milk such asgoat β casein to prepare fusion genes. DNA fragments containing thefusion gene into which the DNA has been inserted are injected into agoat embryo, and the embryo is introduced into a female goat. Thepolypeptide is obtained from the milk produced by the transgenic goatborn to the goat who received the embryo or offsprings thereof. In orderto increase the amount of milk containing the polypeptide produced bythe transgenic goat, hormones may be given to the transgenic goat asappropriate. (Ebert, K. M. et al., Bio/Technology (1994) 12, 699-702).

[0128] As an insect, silkworms may be used. When silkworms are used,baculovirus into which the DNA of interest has been inserted is infectedto the silkworm, and the desired polypeptide can be obtained from thebody fluid of the silkworm (Susumu, M. et al., Nature (1985) 315,592-594).

[0129] Moreover, when plants are used, tabacco, for example, can beused. When tabacco is used, the DNA of interest is inserted into anexpression vector for plants, for example pMON 530, and then the vectoris introduced into a bacterium such as Agrobacterium tumefaciens. Thebacterium is then infected to tabacco such as Nicotiana tabacum toobtain the desired polypeptide from the leaves of the tabacco (Julian,K.-C. Ma et al., Eur. J. Immunol. (1994) 24, 131-138).

[0130] As methods of introducing an expression vector into a host, therecan be used a known method such as the calcium phosphate method(virolgoy (1973) 52, 456-467), the electropolation method (EMBO J.(1982) 1, 841-845), and the like. Considering the frequency of use ofthe host's codon for use in the present invention, a sequence having abetter efficiency of expression can be designed (Grantham, R. et al.,Nucleic Acids Research (1981) 9, r43-r74).

[0131] Gene is introduced as described above into these animals orplants, and polypeptides are produced in the body of the animals or theplants and recovered. Polypeptides expressed and produced as describedabove can be separated from the inside or outside of the host cell andthen may be purified to homogeneity. Separation and purification of theantibody for use in the present invention may be accomplished by, butnot limited to, the separation and the purification methodsconventionally used for protein purification.

[0132] Polypeptides can be separated and purified by selecting andcombining, as appropriate, methods including, but not limited to,chromatography columns such as affinity chromatography, filtration,ultrafiltration, salting-out, dialysis, SDS-polyacrylamide gelelectrophoresis, isoelectric focusing, and the like (Antibodies: ALaboratory Manual, Ed Harlow and David Lane, Cold Spring HarborLaboratory, 1988).

[0133] As chromatography, there may be mentioned, for example, affinitychromatography, ion exchange chromatography, hydrophobic chromatography,gel-filtration, reverse phase chromatography, adsorption chromatography,and the like (Strategies for Protein Purification and Characterization:A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold SpringHarbor Laboratory Press, 1986). These chromatographies can be carriedout using a liquid chromatography such as HPLC and FPLC.

[0134] Polypeptides can be determined using a known method. For example,measurement of absorbance or the Bradford method can be used.

[0135] The present invention provides a method for screening substancesthat inhibit binding between a TAK1 polypeptide and a TAB1 polypeptide,which method comprises contacting the TAB1 polypeptide to the TAK1polypeptide and a test sample, and then detecting or determining theTAK1 polypeptide that is bound to the TAB1 polypeptide; or

[0136] a method for screening substances that inhibit binding between aTAK1 polypeptide and a TAB1 polypeptide, which method comprisescontacting the TAK1 polypeptide to the TAB1 polypeptide and a testsample and then detecting or determining the TAB1 polypeptide that isbound to the TAK1 polypeptide.

[0137] The screening system for use in the present invention may beconducted as an in vitro assay system.

[0138] The in vitro assay system may be conducted in a non-cellularsystem. Specifically, one of the TAB1 polypeptide and the TAK1polypeptide may be previously bound to a support, to which polypeptideare then added the other polypeptide and the test sample, incubated andthen washed followed by detection or determination of binding of thepolypeptide to the other polypeptide bound to the support.Alternatively, the test sample may be added under a homogeneouscondition without binding any of the TAB1 polypeptide and the TAK1polypeptide to the support, incubated, and then immunoprecipitated usingantibody to either of the TAB1 polypeptide and the TAK1 polypeptidefollowed by detection or determination of the amount of the conjugate.

[0139] The TAB1 polypeptide or the TAK1 polypeptide may be a polypeptideproduced by cells that inherently express them, cells into which DNAencoding a polypeptide for use in the present invention has beenintroduced, or animals or plants into which DNA encoding a polypeptidefor use in the present invention has been introduced, which may be usedin a purified or crude-purified form.

[0140] One of the purified or semi-purified TAB1 polypeptide or the TAK1polypeptide is allowed to bind to the support. The polypeptide may beimmobilized onto the support by a standard method in biding thepolypeptides to a support. As supports to which polypeptides are bound,there may be mentioned, for example, insoluble polysaccharides such asagarose, dextran, cellulose, synthetic resin such as polystyrene,polyacrylamide and silicone. More specifically, commercially availablebeads or plates that are produced using the above as a raw material areused. In the case of beads, there may be used columns that are packedwith them. In the case of plates, there may be mentioned multiwellplates (96-well multiwell plates, etc.) or biosensor chips.

[0141] Binding between polypeptides and supports may be effected usingconventionally known methods such as chemical bonding, and physicaladsorption. Alternatively, it is be possible to bind an antibody thatspecifically recognizes the polypeptide to a support in advance so thatthe antibody and the polypeptide become joined. Furthermore,avidin/biotin can also be bound.

[0142] The binding between the TAB1 polypeptide and the TAK1 polypeptidemay be usually effected in buffer solutions. As buffer solutions, forexample, phosphate buffers, Tris buffers and the like may be used.Incubation conditions may be any conditions that are conventionallyused, including the incubation at 4° C. to room temperature for 1 hourto 24 hours. washing after the incubation may be effected in anysolution that does not prevent binding between the TAB1 polypeptide andthe TAK1polypeptide including, for example, a buffer solution containinga surfactant. As surfactants, 0.05% Tween 20 may be used.

[0143] Test specimens to be screened according to the present inventioninclude, for example, peptides, polypeptides, synthetic compounds,microbial fermentation products, marine organism extracts, plantextracts, prokaryotic cell extracts, eukaryotic unicellular extracts, oranimal cell extracts, or libraries thereof. Substances included in thesetest specimens are ones that are expected to act in an inhibitory manneron binding between the TAK1 polypeptide and the TAB1 polypeptide. Theseinhibiting substances inhibit the binding of the TAK1 polypeptide to theTAB1 polypeptide and the binding of the TAB1 polypeptide to the TAK1polypeptide.

[0144] In order to select substances contained in these test specimensthat inhibit the binding of the TAK1 polypeptide to the TAB1 polypeptideand the binding of the TAB1 polypeptide to the TAK1 polypeptide, theyare incubated and washed under an appropriated condition to separate thespecific binding from the non-specific binding. Then the status ofbinding of the polypeptides for use in the present invention can beevaluated.

[0145] In the screening method of the present invention, the controlgroup can be set up together with the group in which the test specimensare contacted to the polypeptides. As the control group, the negativecontrol group having no test specimens, the positive control grouphaving a substance that clearly inhibits binding between the TAB1polypeptide and the TAK1 polypeptide, or both of the groups can be setup.

[0146] When the bound polypeptide is detected or determined inaccordance with the present invention, the bound polypeptide can only bedetected, or the bound polypeptide may be determined in a quantitativemanner. In these cases, the result obtained for the negative controlgroup having no test specimens, the result obtained for the group havinga test specimen, and/or the result obtained for the positive controlgroup having a substance that clearly inhibits binding between the TAB1polypeptide and the TAK1 polypeptide can be compared to detect asubstance that inhibits binding between the TAB1 polypeptide and theTAK1 polypeptide of interest.

[0147] Alternatively, these results may be obtained in numerical values,which values may be compared to determine quantitatively the activity ofthe substance that inhibits binding between the TAB1 polypeptide and theTAK1 polypeptide of interest. When quantitative determinations are made,the numerical value obtained with the negative control group having notest specimens and those obtained with the group in which a testspecimen was applied may be compared to detect the substance thatinhibits binding between the TAB1 polypeptide and the TAK1 polypeptideof interest. The presence of a substance that inhibits binding betweenthe TAB1 polypeptide and the TAK1 polypeptide of interest in the testspecimen would decrease the bound polypeptide, thereby enabling todetermine the specimen that contains the binding-inhibiting substance.

[0148] When quantitative determinations are also made, quantitation maybe made based on a standard curve generated from the numerical valuesobtained with the positive control group containing known amounts of thesubstance that clearly inhibits binding between the TAB1 polypeptide andthe TAK1 polypeptide. When the amount of the bound polypeptide is large,the activity of the substance contained in the test specimen thatinhibits binding between the TAB1 polypeptide and the TAK1 polypeptideis expected to be low, whereas when the amount of the bound polypeptideis small, the activity of the substance contained in the test specimenthat inhibits binding between the TAB1 polypeptide and the TAK1polypeptide is expected to be high.

[0149] In a screening method of a substance that inhibits bindingbetween the TAB1 polypeptide and the TAK1 polypeptide, biosensors may beused that utilize a surface plasmon resonance phenomenon as a means todetect or determine the bound polypeptide. Biosensors that utilize asurface plasmon resonance phenomenon permit a real time observation ofprotein-protein interaction as a surface plasmon resonance signal usingand without labeling a trace amount of protein (for example BIAcore;manufactured by Pharmacia). Thus, by using biosensors such as BIAcore,binding between the TAB1 polypeptide and the TAK1 polypeptide can beevaluated.

[0150] Accordingly, it is intended to contact the TAB1 polypeptide orthe TAK1 polypeptide to a sensor chip on which is immobilized the TAK1polypeptide or the TAB1 polypeptide and then to detect as resonancesignals the TAB1 polypeptide or the TAK1 polypeptide that are bound tothe TAK1 polypeptide or the TAB1 polypeptide.

[0151] Specifically it may be carried out as follows. First a sensorchip CM5 (Biosensor) is activated and then the TAK1 polypeptide or theTAB1 polypeptide is immobilized thereon. Thus, after the sensor chip isactivated with an aqueous solution of EDC/NHS (200 mM EDC(N-ethyl-N′-(3-dimethylaminopropyl)carbonate, hydrochloride), 50 mM NHS(N-hydroxysuccinimide)), it is washed with an HBS buffer (10 mM HEPES,pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.05% Tween 20).

[0152] Then an appropriate amount of TAK1 polypeptide or TAB1polypeptide dissolved in the HBS buffer is contacted to the sensor chipand immobilized thereon. After washing the sensor chip with the HBSbuffer, the active groups remaining on the sensor chip are blocked withan ethanolamine solution (1M ethanolamine hydrochloride, pH 8.5). Thesensor chip is washed again with the HBS buffer for use in the bindingevaluation.

[0153] Then an appropriate amount of TAB1 polypeptide or TAK1polypeptide dissolved in the HBS buffer is injected, whereupon theamount of the TAB1 polypeptide or the TAK1 polypeptide that is. bound tothe TAK1 polypeptide or the TAB1 polypeptide immobilized on the sensorchip is observed as an increment in the value of resonance signal.

[0154] In the above binding-evaluation system, furthermore, a testspecimen is injected after the TAB1 polypeptide or the TAK1 polypeptide.Alternatively, control groups may be set up together with the injectionof the test specimen. As the control groups, the negative control grouphaving no test specimens, the positive control group having a substancethat clearly inhibits binding between the TAB1 polypeptide and the TAK1polypeptide, or both of the groups, can be set up.

[0155] The bound polypeptide is quantitatively determined as a change inthe value of resonance signal. In these cases, the result obtained forthe negative control group having no test specimens, the result obtainedfor the group having a test specimen, and/or the result obtained for thepositive control group having a substance that clearly inhibits bindingbetween the TAB1 polypeptide and the TAK1 polypeptide can be compared todetect and determine a substance that inhibits binding between the TAB1polypeptide and the TAK1 polypeptide of interest.

[0156] As a means of detecting or determining the bound polypeptide inthe method of screening substances that inhibit binding between the TAK1polypeptide and the TAB1 polypeptide of the present invention, either ofthe polypeptides is labeled and the label of the bound polypeptide canbe detected or determined.

[0157] For example, in the above screening method, one polypeptide thatis to be contacted to the other polypeptide together with the testspecimen is labeled beforehand and incubated with the test specimen,washed, and then the bound polypeptide is detected or determined bymeans of the label. Thus, preferably to one polypeptide that has beenbound to a support are contacted the test specimen and the other labeledpolypeptide. After incubating and washing, the label of the boundpolypeptide can be detected or determined.

[0158] The TAK1 polypeptide or the TAB1 polypeptide can be labeled bycommonly known methods. As labels, there may be used, for example,radioisotopes, enzymes, fluorescent substances, biotin/avidin, and thelike. These labels may be commercially available ones. As radioisotopes,there may be mentioned, for example, ³²P, ³³P, ¹³¹I, ¹²⁵I, ³H, ¹⁴C, and³⁵S. As enzymes, there may be mentioned, for example, alkalinephosphatase, horseradish peroxidase, β-galactosidase, β-glucosidase, andthe like. As fluorescent substances, there may be mentioned, forexample, fluorescein isocyanate (FITC) and rhodamine. These arecommercially available and may be labeled by known methods.

[0159] Specifically, the following procedure may be used. Thus, asolution containing one polypeptide is added to a plate, which is thenallowed to stand overnight. After washing the plate, it is blocked with,for example, BSA to prevent non-specific binding of polypeptides. Theplate is washed again, and a test specimen and the other polypeptidethat has been labeled are added to the plate. At the same time, a group(the negative control) containing no test specimens and/or a group (thepositive control) to which a known concentration of a binding-inhibitingsubstance has been added are set up and incubated. After incubation, thewashed and bound polypeptide is detected or determined. For thedetection or determination, in the case of a radioisotope, liquidscintillation is used.

[0160] In the case of an enzyme, a substrate therefor is added and theenzymatic changes, for example color development of the substrate, aredetected or determined. Comparison of these results with the numericalvalue obtained for the control ,group permits the identification of thetest specimen containing the inhibiting substance.

[0161] As a means for detecting or determining the bound polypeptide inthe method of screening substances that inhibit binding between the TAK1polypeptide and the TAB1 polypeptide of the present invention, a primaryantibody that specifically recognizes one polypeptide can be used.

[0162] For example, in the above screening method, to one polypeptideare contacted the other polypeptide together with the test specimen,incubated with the test specimen, washed, and then the bound polypeptideis detected or determined by means of a primary antibody thatspecifically recognizes the polypeptide. Thus, preferably to onepolypeptide that has been bound to a support are contacted the testspecimen and the other polypeptide. After incubating and washing, thebound polypeptide may be detected or determined by means of a primaryantibody that specifically recognizes the polypeptide. Preferably, theprimary antibody has been labeled with a label. The method of producingthe antibody is described below.

[0163] The antibody can be labeled by commonly known methods. As labels,there may be used, for example, radioisotopes, enzymes, fluorescentsubstances, and the like. These labels may be commercially availableones. As radioisotopes, there may be mentioned, for example, ³²P, ³³P,¹³¹I, ¹²⁵I, ³H, ¹⁴C, and ³⁵S. As enzymes, there may be mentioned, forexample, alkaline phosphatase, horseradish peroxidase, β-galactosidase,β-glucosidase, and the like. As fluorescent substances, there may bementioned, for example, fluorescein isocyanate (FITC) and rhodamine.These are commercially available and may be labeled in known methods.

[0164] Specifically, the following procedure may be used. Thus, asolution containing one polypeptide is added to a plate, which is thenallowed to stand overnight. After washing the plate, it is blocked with,for example, BSA to prevent non-specific binding of polypeptides. Theplate is washed again, and a test specimen and the other polypeptide areadded to the plate. At the same time, a group (the negative control)containing no test specimens and/or a group (the positive control) towhich a known concentration of a binding-inhibiting substance has beenadded are set up and incubated.

[0165] After incubation, the plate is washed and an antibody, againstthe polypeptide that was added together with the test specimen, isadded. After an appropriate incubation, the plate is washed and thepolypeptide is detected or determined by means of a primary antibodythat specifically recognizes the polypeptide. For the detection ordetermination, in the case of a radioisotope, liquid scintillation isused. In the case of an enzyme, a substrate therefor is added and theenzymatic changes, for example color development of the substrate, aredetected or determined by means of a photometer. In the case of afluorescent substance, detection and determination may be effected bymeans of a fluorophotometer. Comparison of these results with thenumerical value obtained for the control group permits theidentification of the test specimen containing the inhibiting substance.

[0166] As a means of detecting or determining the bound polypeptide inthe method of screening substances that inhibit binding between the TAK1polypeptide and the TAB1 polypeptide of the present invention, a primaryantibody that specifically recognizes the other peptide or polypeptidefused to the TAB1 polypeptide or the TAK1 polypeptide can be used.

[0167] For example, in the above screening method, to one polypeptideare contacted another polypeptide together with a test specimen,incubated with the test specimen, washed, and then the bound polypeptideis detected or determined by means of a primary antibody thatspecifically recognizes the other peptide or polypeptide fused to thepolypeptide.

[0168] Thus, preferably to one polypeptide that has been bound to asupport are contacted the test specimen and another polypeptide. Afterincubating and washing, the bound polypeptide may be detected ordetermined by means of a primary antibody that specifically recognizesthe other peptide or polypeptide fused to the polypeptide. Preferably,the primary antibody has been labeled with a label. The method ofproducing the antibody is described below.

[0169] The antibody can be labeled by commonly known methods.

[0170] Specifically, the following procedure may be used. Thus, asolution containing one polypeptide is added to a plate, which is thenallowed to stand overnight. After washing the plate, it is blocked with,for example, BSA to prevent non-specific binding of polypeptides. Theplate is washed again, and a test specimen and a polypeptide fused toanother peptide or polypeptide are added to the plate. At the same time,a group (the negative control) containing no test specimens and/or agroup (the positive control) to which a known concentration of abinding-inhibiting substance has been added are set up and incubated.

[0171] After incubation, the plate is washed and an antibody against theother peptide or polypeptide fused to the polypeptide that was addedtogether with the test specimen is added. After an appropriateincubation, the plate is washed and the polypeptide is detected ordetermined by means of a primary antibody that specifically recognizesthe other polypeptide fused to the polypeptide. For the detection ordetermination, in the case of a radioisotope, liquid scintillation isused. In the case of an enzyme, a substrate therefor is added and theenzymatic changes, for example color development of the substrate, aredetected or determined by means of a photometer. In the case of afluorescent substance, detection and determination may be effected bymeans of a fluorophotometer. Comparison of these results with thenumerical value obtained for the control group permits theidentification of the test specimen containing the inhibiting substance.

[0172] As a means of detecting or determining the bound polypeptide inthe method of screening substances that inhibit binding between the TAK1polypeptide and the TAB1 polypeptide of the present invention, a primaryantibody that specifically recognizes the TAB1 polypeptide or the TAK1polypeptide and a secondary antibody that specifically recognizes theprimary antibody can be used.

[0173] For example, in the above screening method, to one polypeptideare contacted another polypeptide together with a test specimen,incubated with the test specimen, washed, and then the bound polypeptideis detected or determined by means of a primary antibody thatspecifically recognizes the polypeptide and a secondary antibody thatspecifically recognizes the primary antibody.

[0174] Thus, preferably to one polypeptide that has been bound to asupport are contacted a test specimen and another polypeptide. Afterincubating and washing, the bound polypeptide may be detected ordetermined by means of a primary antibody that specifically recognizesthe polypeptide and a secondary antibody that specifically recognizesthe primary antibody. Preferably, the secondary Antibody has beenlabeled with a label.

[0175] The method of producing the antibody is described below.

[0176] The antibody can be labeled by commonly known methods.

[0177] Specifically, the following procedure may be used. Thus, asolution containing one polypeptide is added to a plate, which is thenallowed to stand overnight. After washing the plate, it is blocked with,for example, BSA to prevent non-specific binding of polypeptides. Theplate is washed again, and a test specimen and the polypeptide are addedto the plate. At the same time, a group (the negative control)containing no test specimens and/or a group (the positive control) towhich a known concentration of a binding-inhibiting substance has beenadded are set up and incubated.

[0178] After incubation, the plate is washed and a primary antibodyagainst another peptide or polypeptide fused to the polypeptide that wasadded together with the test specimen is added. After an appropriateincubation, the plate is washed and the polypeptide is detected ordetermined by means of the secondary antibody that specificallyrecognizes the primary antibody that specifically recognizes thepolypeptide. For the detection or determination, in the case of aradioisotope, liquid scintillation is used. In the case of an enzyme, asubstrate therefor is added and the enzymatic changes, for example colordevelopment of the substrate, are detected or determined by means of aphotometer. In the case of a fluorescent substance, detection anddetermination may be effected by means of a fluorophotometer. Comparisonof these results with the numerical value obtained for the control grouppermits the identification of the test specimen containing theinhibiting substance.

[0179] As a means of detecting or determining the bound polypeptide inthe method of screening substances that inhibit binding between the TAK1polypeptide and the TAB1 polypeptide of the present invention, a primaryantibody that specifically recognizes the other peptide or polypeptidefused to the TAB1 polypeptide or the TAK1 polypeptide and a secondaryantibody that specifically recognizes the primary antibody can be used.

[0180] For example, in the above screening method, to one polypeptideare contacted another polypeptide together with a test specimen,incubated with the test specimen, washed, and then the bound polypeptideis detected or determined by means of a primary antibody thatspecifically recognizes the other peptide or polypeptide fused to thepolypeptide and a secondary antibody that specifically recognizes theprimary antibody. Thus, preferably, to one polypeptide that has beenbound to a support are contacted the test specimen and the otherpolypeptide. After incubating and washing, the bound polypeptide may bedetected or determined by means of a primary antibody that specificallyrecognizes the other peptide or polypeptide fused to the polypeptide anda secondary antibody that specifically recognizes the primary antibody.Preferably, the secondary antibody has been labeled with a label. Themethod of producing the antibody is described below.

[0181] The antibody can be labeled by commonly known methods.

[0182] Specifically, the following procedure may be used. Thus, asolution containing one polypeptide is added to a plate, which is thenallowed to stand overnight. After washing the plate, it is blocked with,for example, BSA to prevent non-specific binding of polypeptides. Theplate is washed again, and a test specimen and another polypeptide fusedto the other peptide or polypeptide are added to the plate. At the sametime, a group (the negative control) containing no test specimens and/ora group (the positive control) to which a known concentration of abinding-inhibiting substance has been added are set up and incubated.

[0183] After incubation, the plate is washed and a primary antibodyagainst the other peptide or polypeptide fused to the polypeptide thatwas added together with the test specimen is added. After an appropriateincubation, the plate is washed and a secondary antibody thatspecifically recognizes the primary antibody is added. After anappropriate incubation, the plate is washed and the polypeptide isdetected or determined by means of the secondary antibody thatspecifically recognizes the primary antibody that specificallyrecognizes the other polypeptide fused to the polypeptide. For thedetection or determination, in the case of a radioisotope, liquidscintillation is used. In the case of an enzyme, a substrate therefor isadded and the enzymatic changes, for example color development of thesubstrate, are detected or determined by means of a photometer. In thecase of a fluorescent substance, detection and determination may beeffected by means of a fluorophotometer. Comparison of these resultswith the numerical value obtained for the control group permits theidentification of the test specimen containing the inhibiting substance.

[0184] More specifically, the present invention may be conducted by,most specifically, an ELISA (enzyme-linked immunosorbent assay). Thus,the TAK1 polypeptide fused to another peptide or polypeptide, forexample 6×His, is diluted in an immobilization buffer (0.1 M NaHCO₃,0.02% NaN₃, pH 9.6). A suitable amount of an aqueous solution that wasdiluted is added to each well of a 96-well immunoplate (manufactured byNunc), which is then incubated overnight at 4° C.

[0185] After each well is washed three times with the wash buffer(prepared to 0.05% Tween 20 in PBS), 200 μl of a 5% solution of BSA(manufactured by SIGMA) dissolved in PBS is added to block overnight at4° C.

[0186] Then, each well is washed three times with the wash buffer, andappropriate amounts of the TAB1 polypeptide fused to another peptide orpolypeptide, for example FALG, and a test specimen are added thereto andincubated at room temperature for one hour. Each well is washed threetimes with the wash buffer, and 100 μl of mouse anti-FLAG M2 antibody(manufactured by IBI) dissolved to 3 mg/ml in a dilution buffer is addedto each well and incubated at room temperature for one hour.

[0187] Each well is washed three times with a wash buffer, and 100 μl ofalkaline phosphatase-labeled goat anti-mouse IgG antibody (manufacturedby ZYMED) diluted 1000-fold in the dilution buffer is added to each welland incubated at room temperature for one hour. Each well is washed fivetimes with the wash buffer, and 100 μl of the color development solution(substrate buffer; p-nitrophenyl phosphate dissolved to 1 mg/ml in 50 mMNaHCO₃, 10 mM MgCl₂, pH 9,8, manufactured by Sigma) is added to eachwell and incubated at room temperature. Subsequently, absorbance at 405nm is measured using a microplate reader (Model 3550, manufactured byBIO-RAD). Comparison of these results with the numerical value obtainedfor the negative control group and/or positive control group permits theidentification of the test specimen containing the inhibiting substance.

[0188] The screening method of the present invention may also be usedfor the High Throughput Screening (HTS). Specifically, steps up to theblocking may be conducted manually, and the subsequent reactions can beautomated by robotization to realize High Throughput Screening.

[0189] Thus, the TAK1 polypeptide fused to another peptide orpolypeptide, for example 6×His, is diluted in the immobilization buffer(0.1 M NaHCO₃, 0.02% NaN₃, pH 9.6). A suitable amount of the aqueoussolution that was diluted to each well of a 96-well immunoplate(manufactured by Nunc) is added and then incubated overnight at 4° C.

[0190] After each well is washed three times with the wash buffer(prepared to 0.05% Tween 20 in PBS), 200 μl of a 5% solution of BSA(manufactured by SIGMA) dissolved in PBS is added to block overnight at4° C.

[0191] Subsequently, an immunoplate after blocking is mounted to, forexample, the Biomek 2000 HTS system (manufactured by Beckman) and thecontrol program of the system is executed. At this time the delivery ofthe solution to each well of the immunoplate and the removal thereof canbe carried out using the Biomek 2000 HTS system (manufactured byBeckman) and the Multipipette 96-well simultaneous dispenser(manufactured by Sagian) as a dispensing instrument. Washing of eachwell of the immunoplate can also be carried out using the EL404microplate washer (Bio Tek). Measurement of absorbance can be made usingthe SPECTRAmax250 plate reader (manufactured by Molecular Devices).

[0192] The program is set so as to perform the following steps. Thus,each well is washed three times with the wash buffer, appropriateamounts of the test specimen and the other peptide or polypeptidediluted in the dilution buffer (1% BSA, 0.5% Tween 20, PBS) such as theTAB polypeptide fused to MBP (maltose-binding protein) are added. At thesame time, a group (the negative control) containing no test specimensand a group (the positive control) to which a known concentration of abinding-inhibiting substance has been added are set up and incubated atroom temperature for one hour.

[0193] Each well is washed three times with the wash buffer, 100 μl ofrabbit anti-MBP antiserum (manufactured by New England Biolabs) is addedto each well, and incubated at room temperature for one hour. Each wellis washed three times with the wash buffer, 100 μl of alkalinephosphatase-labeled goat anti-mouse IgG antibody (manufactured by TAGO)diluted 5000-fold in the dilution buffer is added to each well, andincubated at room temperature for one hour.

[0194] Each well is then washed five times with the wash buffer, 100 μlof the color development solution (substrate buffer; p-nitrophenylphosphate dissolved to 1 mg/ml in 50 mM NaHCO₃10 mM MgCl₂, pH 9,8,manufactured by sigma) is added to each well, and incubated at roomtemperature. Subsequently, absorbance at 405 nm is measured using amicroplate reader, the Biomek plate reader (manufactured byBeckman/Molecular Devices). Comparison of these results with thenumerical value obtained for the control group permits theidentification of the test specimen containing the inhibiting substance.

[0195] Antibodies for use in the present invention may be those that arecommercially available or that are contained in commercially availablekits, or they can be obtained as monoclonal or polyclonal antibodiesusing known methods.

[0196] Monoclonal antibodies can be obtained by using the desiredsensitizing antigen, which is immunized in a conventional method forimmunization, by fusing the immune cells thus obtained with known parentcells, and screening monoclonal antibody-producing cells using a knownscreening method.

[0197] Specifically, monoclonal or polyclonal antibodies may begenerated as follows.

[0198] Though the sensitizing antigen for generation of antibodies isnot limited by the animal species from which the antibodies areobtained, it is preferably derived from a mammal from which peptides orpolypeptides actually used in the present invention are derived, such ashumans, mice, or rats. Among them, sensitizing antigens derived fromhumans are preferred. When, for example, a human TAB1 polypeptide orhuman TAK1 polypeptide is used as the sensitizing antigen, thenucleotide sequence and the amino acid sequence thereof can be obtainedusing the gene sequence disclosed in the present invention. Furthermore,when other peptides or polypeptides that are fused with the human TAB1polypeptide or human TAK1 polypeptide are used as the sensitizingantigen, the peptides and the polypeptides can be chemically synthesizedor can be obtained using genetic engineering technology.

[0199] Peptides or polypeptides that are used as the sensitizing antigenmay be full-length or fragments thereof. As fragments, for example,C-terminal fragments or N-terminal fragments may be mentioned.

[0200] Mammals to be immunized with the sensitizing antigen are notspecifically limited, and they are preferably selected in considerationof their compatibility with the parent cell for use in cell fusion. Theygenerally include rodents, lagomorphs, and primates.

[0201] Rodents include, for example, mice, rats, hamsters, and the like.Lagomorphs include, for example, rabbits. Primates include, for example,monkeys. As monkeys, catarrhines (Old-World monkeys) such as cynomolgi(crab-eating macaque), rhesus monkeys, sacred baboons, chimpanzees etc.are used.

[0202] Immunization of animals with a sensitizing antigen is carried outusing a known method. A general method, for example, involves theintraperitoneal or subcutaneous administration of a sensitizing antigento the mammal. Specifically, a sensitizing antigen which has beendiluted and suspended in an appropriate amount of phosphate bufferedsaline (PBS) or physiological saline etc. is mixed, as desired, with anappropriate amount of a conventional adjuvant, for example Freund'scomplete adjuvant. After being emulsified, it is preferably administeredto the mammal for several times every 4 to 21 days. Alternatively asuitable carrier may be used at the time of immunization of thesensitizing antigen. After such immunization, the increase in thedesired antibody levels in the serum is confirmed by a conventionalmethod.

[0203] In order to obtain polyclonal antibodies, the blood of the mammalthat was sensitized with the antigen is removed after the increase inthe desired antibody levels in the serum has been confirmed. Serum isseparated from the blood. As polyclonal antibodies, serum containing thepolyclonal antibodies may be used, or, as desired, the tractioncontaining the polyclonal antibodies may be isolated from the serum.

[0204] In order to obtain monoclonal antibodies, immune cells of themammal that was sensitized with the antigen are removed and aresubjected to cell fusion after the increase in the desired antibodylevels in the serum has been confirmed. At this time preferred immunecells that are subjected to cell fusion include, in particular, thespleen cell.

[0205] The mammalian myeloma cells as other parent cells which aresubjected to cell fusion with the above-mentioned immune cellspreferably include various known cell lines such as P3 (P3X63Ag8.653)(Kearney, J. F. et al., J. Immunol. (1979) 123: 1548-1550), P3X63Ag8.U1(Yelton, D. E., et al., Current Topics in Microbiology and Immunology(1978) 81: 1-7), NS-1 (Kohler, G. and Milstein, C., Eur. J. Immunol.(1976) 6: 511-519), MPC-11 (Margulies, D. H. et al., Cell (1976) 8:405-415), SP2/0 (Shulman, M. et al., Nature (1978) 276: 269-270), FO (deSt. Groth, S. F. and Scheidegger, D., J. Immunol. Methods (1980) 35:1-21), S194 (Trowbridge, I. S., J. Exp. Med. (1978) 148: 313-323), R210(Galfre, G. et al., Nature (1979) 277: 131-133) and the like.

[0206] Cell fusion between the above immune cells and the myeloma cellsmay be essentially conducted in accordance with a known method such asis described in Milstein et al. (Kohler, G. and Milstein, C., MethodsEnzymol. (1981) 73: 3-46) and the like.

[0207] More specifically, the above cell fusion is carried out in theconventional nutrient broth in the presence of, for example, a cellfusion accelerator. As the cell fusion accelerator, for example,polyethylene glycol (PEG), Sendai virus (HVJ) and the like may be used,and, in addition, an adjuvant such as dimethyl sulfoxide etc. may beadded as desired to enhance efficiency of the fusion.

[0208] The preferred ratio of the immune cells and the myeloma cells tobe used is, for example, 1 to 10 times more immune cells than themyeloma cells. Examples of culture media to be used for the above cellfusion include RPMI1640 medium and MEM culture medium suitable for thegrowth of the above myeloma cell lines, and the conventional culturemedium used for this type of cell culture, and besides a serumsupplement such as fetal calf serum (FCS) may be added.

[0209] In cell fusion, predetermined amounts of the above immune cellsand the myeloma cells are mixed well in the above culture liquid, towhich a PEG solution previously heated to about 37° C., for example aPEG solution with a mean molecular weight of about 1000 to 6000, isadded at a concentration of 30 to 60% (w/v), and mixed to obtain thedesired fusion cells (hybridomas). Then by repeating the sequentialaddition of a suitable culture liquid and centrifugation to remove thesupernatant, cell fusion agents etc. which are undesirable for thegrowth of the hybridoma can be removed.

[0210] Said hybridoma is selected by culturing in a conventionalselection medium, for example, the HAT culture medium (a culture liquidcontaining hypoxanthine, aminopterin, and thymidine). Culturing in saidHAT culture medium is continued generally for a period of timesufficient to effect killing of the cells other than the desiredhybridoma (non-fusion cells), generally several days to several weeks.Then, the conventional limiting dilution method is conducted in whichthe hybridomas that produce the desired antibody are screened andcloned.

[0211] In addition to obtaining the above hybridoma by immunizing ananimal other than the human with an antigen, it is also possible tosensitize human lymphocytes infected with EB virus with a peptide orpolypeptide, cells expressing them, or their lysates in vitro, and toallow the resulting sensitized lymphocytes to be fused with ahuman-derived myeloma cell having a permanent division potential, forexample U266, and thereby to obtain a hybridoma producing the desiredhuman antibody having the activity of binding the peptide or thepolypeptide (see Japanese Unexamined Patent Publication (Kokai) No.63(1988)-17688).

[0212] Furthermore, a transgenic animal having a repertoire of humanantibody genes is immunized with the antigen peptide or polypeptide,cells expressing them or lysates thereof to obtain theantibody-producing cells, which are used to obtain human antibodyagainst the peptide or polypeptide for use in the present inventionusing hybrodomas fused to myeloma cells (see International PatentApplication WO 92-03918, WO 93-2227, WO 94-02602, WO 94-25585, WO96-33735 and WO 96-34096).

[0213] The monoclonal antibody-producing hybridomas thus constructed canbe subcultured in the conventional culture liquid, or can be stored fora prolonged period of time in liquid nitrogen.

[0214] In order to obtain monoclonal antibodies from said hybridoma,there may be employed a method in which said hybridoma is cultured inthe conventional method and the antibodies are obtained as the culturesupernatant, or a method in which the hybridoma is administered to andgrown in a mammal compatible with said hybridoma and the antibodies areobtained as the ascites. The former method is suitable for obtaininghigh-purity antibodies, whereas the latter is suitable for a large scaleproduction of antibodies.

[0215] In addition to using a hybridoma to produce antibody, immunecells that produce the desired antibody, for example the sensitizedlymphocytes that have been immortalized with an oncogene, may be used toobtain the antibody.

[0216] A monoclonal antibody thus produced can also be obtained as arecombinant antibody by recombinant gene technology. For example, anantibody gene may be cloned from the hybridoma or an immune cell such assensitized lymphocytes that produce antibodies, and is integrated into asuitable vector which is then introduced into a host to produce saidantibody. Recombinant antibodies may also be used in the presentinvention (see, for example, Borrebaeck, C. A. K., and Larrick, J. W.,THERAPEUTIC MONOCLONAL ANTIBODIES, published in the United Kingdom byMACMILLAN PUBLISHERS LTD. 1990).

[0217] Antibodies for use in the present invention may be antibodyfragments or modified versions thereof as long as they have the desiredbinding activity. For example, as fragments of antibody, there may bementioned Fab, F(ab′)₂, Fv or single-chain Fv (scFv) in which Fv or Fv'sof the H chain and the L chain were ligated via a suitable linker.Specifically antibodies are treated with an enzyme such as papain orpepsin, to produce antibody fragments, or genes encoding these antibodyfragments are constructed and then introduced into an expression vector,which is expressed in a suitable host cell (see, for example, Co, M. S.et al., J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, A.H., Methods in Enzymology (1989) 178, 476-496; Plucktrun, A. and Skerra,A., Methods in Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods inEnzymol. (1986) 121, 652-663; Rousseaux, J. et al., Methods in Enzymol.(1986) 121, 663-669; Bird, R. E. and Walker, B. W., Trends Biotechnol.(1991) 9, 132-137).

[0218] Antibodies produced and expressed as described above can beseparated from the inside or outside of the host cell and then may bepurified to homogeneity. Separation and purification of the antibody foruse in the present invention may be accomplished by, but is not limitedto, the separation and the purification methods conventionally used forproteins.

[0219] These methods include chromatography columns such as affinitychromatography, filtration, ultrafiltration, salting-out, dialysis, SDSpolyacrylamide gel electrophoresis, isoelectric focusing, and the like,from which methods can be selected and combined as appropriate forseparation and purification of antibodies (Antibodies: A LaboratoryManual, Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988).

[0220] As columns for use in affinity chromatography, there can bementioned Protein A column and Protein G column. Examples of thecarriers used in the Protein A column are Hyper D, POROS, Sepharose F.F. (Pharmacia) and the like.

[0221] As chromatography other than the above-mentioned affinitychromatography, there can be mentioned, for example, ion exchangechromatography, hydrophobic chromatography, gel-filtration, reversephase chromatography, adsorption chromatography, and the like(Strategies for Protein Purification and Characterization: A LaboratoryCourse Manual, Ed Daniel R. Marshak et al., Cold Spring HarborLaboratory Press, 1986). These chromatographies can be carried out usinga liquid chromatography such as HPLC and FPLC.

[0222] The concentration measurement and activity confirmation of theantibody obtained as above can be made by known methods such asenzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), orfluorescent antibody assay.

[0223] Substances that inhibit binding between the TAB1 polypeptide andthe TAK1 polypeptide that were obtained using the screening method ofthe present invention can be obtained by screening the test compoundssuch as peptides, proteins, non-peptide compounds, synthetic compounds,microbial fermentation products, marine organism extracts, plantextracts, cell extracts, or animal cell extracts by screening methods.These test compounds may be novel compounds, or existing compounds.

[0224] These binding-inhibiting substances are compounds that inhibitbinding between the TAB1 polypeptide and the TAK1 polypeptide. Compoundsthat were changed by addition, deletion, or substitution of part of thestructure of substances that inhibit binding between the TAB1polypeptide and the TAK1 polypeptide obtained by the screening method ofthe present invention are included in the substances that inhibitbinding between the TAB1 polypeptide and the TAK1 polypeptide obtainedby the screening method of the present invention.

[0225] Substances that inhibit binding between the TAB1 polypeptide andthe TAK1 polypeptide obtained by the screening method of the presentinvention may be substances that activate signal transduction of TGF-βor substances that suppress signal transduction of TGF-β. TGF-β is knownto have the effect of enhancing extracellular matrix protein production,inhibiting cellular growth, causing monocyte migration, inducingbiologically active substances, suppressing immunity, depositing amyloidβ protein, and the like. Both of the TAB1 polypeptide and the TAK1-polypeptide are responsible for signal transduction of TGF-β each bybinding thereto. Thus, substances that inhibit binding between the TAB1polypeptide and the TAK1 polypeptide obtained by the screening method ofthe present invention can be obtained as a substance that activates orsuppresses signal transduction of TGF-β.

[0226] When substances that inhibit binding between the TAB1 polypeptideand the TAK1 polypeptide obtained by the screening method of the presentinvention are used as medicaments for humans and mammals such as mice,rats, guinea pigs, rabbits, chickens, cats, dogs, sheep, pigs, cattle,monkeys, baboons, and chimpanzees, they may be used in the conventionalmethod.

[0227] For example, they may be used, as desired, orally as capsules andmicrocapsules, or parenterally in the form of sterile solutions withwater or other pharmaceutically acceptable liquids or suspensions. Forexample, substances that inhibit binding between the TAB1 polypeptideand the TAK1 polypeptide are produced in unit dosage forms required forgenerally accepted formulations by mixing with pharmaceuticallyacceptable carriers, excipients, vehicles, antiseptics, stabilizers, andadhesion inhibitors. The amount of active ingredients in theseformulations is designed to provide an indicated suitable range ofdoses. As additives that may be blended for tables or capsules, forexample, gelatin, HSA (human serum albumin), crystalline cellulose,alginic acid, magnesium stearate, sucrose, and lactose may be used.

[0228] As aqueous solutions for injection, there may be mentioned, forexample, isotonic liquids such as physiological saline, glucose andother adjuvants such as D-sorbitol, D-mannose, D-mannitol, and sodiumchloride, and they may be used in combination with suitable solubilizingagents such as alcohols, specifically ethanol, polyalchohols including,for example, propylene glycol and polyethylene glycol, nonionicsurfactants such as polysorbate 80TM, HCO-50, benzyl benzoate, phosphatebuffer, sodium acetate buffer, procaine hydrochloride, benzyl alchohol,and phenol.

[0229] The dosage of substances that inhibit binding between the TAB1polypeptide and the TAK1 polypeptide for a human adult (assuming thebody weight of 60 kg) is, when given orally, usually about 0.1 to 100mg/day, preferably about 1.0 to 50 mg/day, and more preferably about 1.0to 20 mg/day, though this may vary depending on the medical conditions.

[0230] When given parenterally, the dose per administration for a humanadult (assuming the body weight of 60 kg) of usually about 0.01 to 30mg/day, preferably about 0.1 to 20 mg/day, and more preferably about 0.1to 10 mg/day in the case of injections is conveniently administered viaintravenous injection, though this may vary depending on the subjectorgan, medical conditions, and the method of administration. For otheranimals, the amount converted in terms of the body weight of 60 kg maybe administered.

EXAMPLES

[0231] The present invention will now be explained in more details withreference to the examples. It should be noted, however, that the presentinvention is not limited to them in any way.

Example 1 Construction of a Baculovirus Transfer Vector for RecombinantHuman TAB1 and Recombinant Human TAK1

[0232] In order to express a full-length human TAB1 polypeptide and afull-length human TAK1 polypeptide by a baculovirus expression system, abaculovirus transfer vector was constructed. At this time, it wasdesigned to add a peptide tag in order to facilitate purification anddetection.

[0233] Thus, a FLAG tag comprising 8 amino acids(Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys; SEQ ID NO: 5) was added to the carboxyterminal of human TAB1. Also, a 6×His tag (Janknecht, R. et al., Gene(1992) 121, 321-324) comprising 6 contiguous His residues(His-His-His-His-His-His; SEQ ID NO: 6) was added to the carboxyterminal of human TAK1. Each recombinant polypeptide is expressed as afusion polypeptide, human TAB1-FLAG or human TAK1-6×His.

[0234] In order to obtain a DNA fragment encoding human TAB1-FLAG, a PCRmethod was carried out with plasmid pBS-TAB1 (Shibuya, H. et al.,Science (1996) 272, 1179-1182) as a template using a sense primer TABFS(SEQ ID NO: 7) and an antisense primer TAB1AS (SEQ ID NO: 8) that weresynthesized using a primer synthesizer.

[0235] The sense primer TABFS comprises a nucleotide sequence fromnucleotide A at position 30 to nucleotide G at position 47 of the regionencoding a full-length human TAB1 polypeptide contained in plasmidPBS-TAB1 as set forth in SEQ ID NO: 1 or 2 after the recognition site ofthe restriction enzyme EcoRI. The antisense primer TAB1AS comprises anucleotide sequence complementary to a series of nucleotide sequencescomprising a nucleotide sequence encoding 5 amino acid sequencecomprised of Gly-Thr-Gly-Gly-Ser (SEQ ID NO: 9), a nucleotide sequenceencoding the FLAG tag, two stop codons, and a recognition site of therestriction enzyme XbaI, after a nucleotide sequence from nucleotide Aat position 1524 to nucleotide G at position 1541 of the region encodingthe human TAB1 polypeptide contained in the plasmid pBS-TAB1 as setforth in SEQ ID NO: 3 or 4 (FIG. 1).

[0236] The PCR method comprised a total of 25 cycles of 94° C. for 1minute, 55° C. for 1 minute, and 72° C. for 2 minutes per cycle. PCRreaction products were separated and purified by a 1% low-melting pointagarose gel (manufactured by Sigma), digested with the restrictionenzymes EcoRI and XbaI, and then were inserted into the baculovirustransfer vector pBacPAK9 (manufactured by CLONTECH).

[0237] The nucleotide sequence of the inserted DNA fragment wasdetermined by a DNA sequencer (Model 373A, manufactured by ABI) therebyto confirm that the correct nucleotide sequence had bee inserted.

[0238] The plasmid comprising a nucleotide sequence encoding humanTAB1-FLAG was designated as pBacTABF. The nucleotide sequence and theamino acid sequence encoding human TAB1-FLAG are shown in SEQ ID NO: 10and 11.

[0239] In order to obtain a DNA fragment encoding human TAK1-6×His, aPCR method was carried out with plasmid phTAK1 (Japanese UnexaminedPatent Publication (Kokai) No. 9(1997)-163990) as a template using asense primer TAKS (SEQ ID NO: 12) and an antisense primer TAKAS (SEQ IDNO: 13) that were synthesized using a primer synthesizer.

[0240] The sense primer TAKS comprises a nucleotide sequence fromnucleotide A at position 183 to nucleotide C at position 200 of theregion encoding a human TAK1 polypeptide contained in plasmid phTAK1 asset forth in SEQ ID NO: 3 after the recognition site of the restrictionenzyme EcoRI. The antisense primer TAKAS comprises a nucleotide sequencecomplementary to a nucleotide sequence encoding 5 amino acid sequencecomprised of Gly-Thr-Gly-Gly-Ser, a nucleotide sequence encoding the6×His tag, two stop codons, and a recognition site of the restrictionenzyme XbaI, after a nucleotide sequence from nucleotide A at position1902 to nucleotide A at position 1919 of the region encoding the humanTAK1 polypeptide contained in plasmid phTAK1 (FIG. 1).

[0241] The PCR method comprised a total of 25 cycles of 94° C. for 1minute, 55° C. for 1 minute, and 72° C. for 2 minutes per cycle. PCRreaction products were separated and purified by a 1% low-melting pointagarose gel (manufactured by Sigma), digested with the restrictionenzymes EcoRI and XbaI, and then were inserted into the baculovirustransfer vector pBacPAK9 (manufactured by CLONTECH). The nucleotidesequence of the inserted DNA fragment was determined by a DNA sequencer(Model 373A, manufactured by ABI) thereby to confirm that the correctnucleotide sequence had been inserted. The plasmid comprising anucleotide sequence encoding human TAK1-6×His was designated aspBacTAKF. The nucleotide sequence and the amino acid sequence encodinghuman TAK1-6×His are shown in SEQ ID NO: 14 and 15.

Example 2 Expression of Recombinant Human TAB1-FLAG and Human TAK1-6×HisPolypeptides Example 2-1 Construction of a Recombinant Baculovirus

[0242] A recombinant baculovirus was constructed in accordance with theinstructions by CLONTECH.

[0243] Thus, for the construction of the recombinant human TAB1-FLAGbaculovirus, 0.5 μg of the transfer vector pBacTABF of the above Example1, 5 μl of the Bsu36 I digest of baculovirus BacPAK6 DNA (manufacturedby CLONTECH), and 50 μl of a 0.1 mg/ml lipofectin solution (manufacturedby CLONTECH) were diluted to a total of 100 μl in distilled water. Theaqueous solution was mixed with 1.5 ml of serum-free medium and wasadded with 1×10⁶ lined insect cells Sf9 (ATCC CRL 1711).

[0244] At 5 hours after the addition, 1.5 ml of the insect cell culturemedium (containing a medium supplement for the Grace insect cells,manufactured by GIBCO BRL) supplemented with 10% fetal bovine serum wasfurther added, incubated at 27° C. for 5 days, and then the supernatantwas recovered. Using the culture supernatant, a plaque assay was carriedout according to the instructions by the manufacturer to isolate arecombinant baculovirus that expresses recombinant TAB1-FLAG from asingle plaque.

[0245] Using the transfer vector pBacTAKF described in the above WorkingExample 1 in a similar procedure, a recombinant baculovirus expressingthe recombinant human TAK1-6×His was constructed.

Example 2-2 Expression of each Recombinant Polypeptide Using aRecombinant Baculovirus

[0246] A 100 M.O.I. amount of a recombinant baculovirus was infected to1×10⁹ lined insect cells Sf9, and was incubated in 1000 ml of an insectcell culture medium supplemented with 2% fetal bovine serum at 27° C.for 5 days. Cells after incubation were washed three times in PBS(Dulbecco PBS, manufactured by Nissui), and were subjected to thefollowing purification.

Example 3 Purification of Recombinant Human TAB1-FLAG and HumanTAK1-6×His Polypeptide Example 3-1 Purification of the Recombinant HumanTAB1-FLAG Polypeptide

[0247] (1) Preparation of a Cleared Lysate

[0248] After the cells obtained in the above 2-2 were suspended in theTMN buffer (20 mM Tris-HCl, 3 mM MgCl₂, 150 mM NaCl, 0.1 mg/ml PMSF, 1μg/ml leupeptin, 1 μg/ml aprotinin, pH 7.5) to a density of 4×10⁷cells/ml, the cells were treated by an ultrasonic disrupting instrument(SONIFIER 250, manufactured by BRANSON) until 90% of the cells weredisrupted. The insoluble material in the disrupted solution wasprecipitated by centrifuging by a centrifuge (model MRX-150,manufactured by TOMY) at 14,000 rpm for 10 minutes. The supernatant thusobtained was filtered using a 0.45 μm filter (SterivexTM-HV,manufactured by MILLIOPORE), and the filtrate was used as the clearedlysate.

[0249] (2) Purification with an Anti-FLAG M2 Affinity Gel

[0250] In order to purify the recombinant human TAB1-FLAG polypeptidefrom the cleared lysate, affinity purification with anti-FLAG antibodywas carried out as follows:

[0251] Two ml of the anti-FLAG antibody M2 affinity gel (manufactured byIBI) was equilibrated with a TBS buffer (50 mM Tris-HCl, 150 mM NaCl, pH7.4) in the bed support (Poly-Prep Chromatography Column, manufacturedby BIO-RAD). The above supernatant was added to the equilibratedanti-FLAG antibody M2 affinity gel, and the human TAB1-FLAG polypeptidewas bound thereto. The column was washed with 30 ml of the TBS buffer,and the bound polypeptide was eluted with 2 ml each of the elutionbuffer (0.1 M glycine-HCl, pH 3.5) in six portions.

[0252] After the elution, the elution buffer was replaced with PBS usingthe gel filtration column PD-10 (manufactured by Pharmacia), which wasused as a purified product of the recombinant human TAB1-FLAGpolypeptide. The purity of the recombinant human TAB1-FLAG polypeptidewas measured by the BCA* Protein Assay Reagent (manufactured by PIERCE)using BSA as a standard.

Example 3-2 Purification of the Recombinant Human TAK1-6×His Polypeptide

[0253] (1) Preparation of the Cleared Lysate

[0254] The cells obtained in the above 2-2 were suspended in thesonication buffer (20 mM Tris-HCl, 100 NaCl, 0.1 mg/ml PMSF, 1 μg/mlleupeptin, 1 μg/ml aprotinin, pH 8.0) to a density of 4×10⁷ cells/ml,and then the cells were treated by an ultrasonic disrupting instrument(SONIFIER 250, manufactured by BRANSON) until 90% of the cells weredisrupted. The insoluble material in the disrupted solution wasprecipitated by centrifuging by a centrifuge (model MRX-150,manufactured by TOMY) at 14000 rpm for 10 minutes. The supernatant thusobtained was filtered using a 0.45 μm filter (Sterivex™-HV, manufacturedby MILLIOPORE), and the filtrate was used as the cleared lysate.

[0255] (2) Purification with TALON™ Metal Affinity Resin

[0256] In order to purify the recombinant human TAK1-6×His polypeptidefrom the cleared lysate, purification with affinity resin (TALON™ MetalAffinity Resin, manufactured by CLON TECH) was carried out as follows:

[0257] Two ml of the affinity resin equilibrated with the sonicationbuffer and the cleared lysate of the above Working Example 3-2 (1) weremixed under shaking for 20 minutes, to which the recombinant humanTAK1-6×His polypeptide was ligated. After removing the supernatant bycentrifugation at 700 ×g, the affinity resin was mixed under shaking in20 ml of the wash buffer (10 mM imidazole, 20 mM Tris-HCl, 100 mM NaCl,pH 8.0) at 4° C. for 10 minutes, followed by the removal of thesupernatant by centrifugation at 700 ×g.

[0258] After the affinity resin was mixed under shaking in 20 ml of thewash buffer (10 mM imidazole, 20 mM Tris-HCl, 100 mM NaCl, pH 8.0) at 4°C. for 10 minutes, the resin was washed by removing the supernatant bycentrifugation at 700 ×g. The affinity resin after washing was suspendedin 2 ml of the wash buffer and was transferred to the bed support(Poly-Prep Chromatography Column, manufactured by BIO-RAD), and theaffinity resin was further washed with 6 ml of the wash buffer.

[0259] The affinity resin was subjected to elution by the elution buffer(50 mM imidazole, 20 mM Tris-HCl, 100 mM NaCl, pH 8.0) in six portionsof 2 ml each. After elution, the elution buffer was replaced with PBSusing the gel filtration column PD-10 (manufactured by Pharmacia), whichwas used as a purified product of the recombinant human TAK1-6×Hispolypeptide. The purity of the recombinant human TAK1-6×His polypeptidewas measured by the BCA* Protein Assay Reagent (manufactured by PIERCE)using BSA as a standard.

Example 4 Preparation of the Recombinant MBP-TAB1C-FLAG Example 4-1Construction of the Expression Vector

[0260] By an expression system using Escherichia coli (E. coli), anexpression vector for expression in E. coli of a fusion polypeptide of apolypeptide comprising 81 amino acids at the carboxy terminal of thehuman TAB1 polypeptide and maltose-binding protein was constructed.

[0261] At this time, a FLAG tag was added to the carboxy terminal of theabove fusion polypeptide in order to facilitate purification anddetection.

[0262] In order to obtain a DNA fragment encoding 81 amino acids at thecarboxy terminal of the human TAB1 and the FLAG tag, a PCR method wascarried out with plasmid pBacTABF as a template using a sense primerTABC1 (SEQ ID NO: 16) and an antisense primer TABC3 (SEQ ID NO: 17) thatwere synthesized using a primer synthesizer.

[0263] The sense primer TABC1 comprises a nucleotide sequence fromnucleotide C at position 1281 to nucleotide T at position 1307 of theregion encoding the human TAB1 polypeptide contained in plasmid pBacTABFas set forth in SEQ ID NO: 9 after the recognition site of therestriction enzyme XmnI.

[0264] The antisense primer TABLAS comprises a nucleotide sequencecomplementary to a series of nucleotide sequences comprising anucleotide sequence encoding a nucleotide sequence encoding the FLAGtag, two stop codons, and a recognition site of the restriction enzymeHindIII, after a nucleotide sequence from nucleotide C at position 1489to nucleotide G at position 1518 of the region encoding the human TAB1polypeptide contained in the plasmid pBacTABF as set forth in SEQ ID NO:9.

[0265] The PCR method comprised a total of 25 cycles of 94° C. for 1minute, 55° C. for 1 minute, and 72° C. for 1 minute per cycle. PCRreaction products were separated and purified by a 1% low-melting pointagarose gel (manufactured by Sigma), digested with the restrictionenzymes XmnI and HindIII, and then were inserted into a fusionpolypeptide expression vector pMAL-p2, (manufactured by New EnglandBiolabs).

Example 4-2 Expression of the Fusion Polypeptide

[0266] A fusion polypeptide was expressed according. to the instructionsby New England Biolabs. Thus, E. coli having the plasmid obtained asabove was grown overnight and 2 ml thereof was inoculated into 200 ml ofa rich broth (10 g tryptone, 5 g yeast extract, 5 g NaCl, 2 g glucose,100 mg ampicillin/liter). It was incubated under shaking at 37° C. tillthe cell density reached A₆₀₀=1, at which time IPTG(isopropylthiogalactoside) was added to a final concentration of 0.3 mM.It was further incubated under shaking at 37° C. for 2 hours and thenwas centrifuged at 4000 ×g for 10 minutes to harvest the cells.

Example 4-3 Preparation of the Periplasm Fraction

[0267] The cells collected in the above Example 4-2 was resuspended in25 ml of 30 mM Tris, 20% sucrose, pH 8.0, and 50 μl of 0.5 M EDTA, pH8.0, was added thereto followed by incubation under shaking for 10minutes. Subsequently, it was cetrifuged at 8000 ×g to remove thesupernatant and the cells were resuspended in 25 ml of ice-cold 5 mMMgSO₄ and was further incubated under shaking in the ice. Aftercentrifugation at 8000 ×g, the supernatant was recovered as theperiplasm fraction.

Example 4-4 Purification with the Anti-FLAG M2 Affinity Gel

[0268] In order to purify recombinant MBP-TAB1C-FLAG polypeptide fromthe periplasm fraction, affinity purification with anti-FLAG M2 antibodywas carried out in a similar manner to Example 3-1 (2) to obtain apurified product of the recombinant MBP-TAB1C-FLAG polypeptide.

Example 5 Construction of an ELISA System Using the Purified Product

[0269] Using the purified product obtained as above an ELISA system wasconstructed to detect in vitro an interaction between the recombinanthuman TAB1 polypeptide or the recombinant MBP-TAB1C-FLAG polypeptide andthe recombinant human TAK1 polypeptide. In the ELISA system it isintended to contact the recombinant human TAB1-FLAG polypeptide or thehuman MBP-TAB1C-FLAG polypeptide to a 96-well immunoplate to which thehuman TAK1-6×His polypeptide had been previously immobilized and therebyto detect the recombinant human TAB1-FLAG polypeptide or the recombinantMBP-TAB1C-FLAG polypeptide using a primary and secondary antibody.

Example 5-1 Construction of an in Vitro Binding-Evaluation System

[0270] (1) The purified product of human TAK1-6×His polypeptide wasdiluted in the immobilization buffer (0.1 M NaHCO₃, 0.02% NaN₃, pH 9.6).To each well of a 96-well immunoplate (manufactured by Nunc) was added100 μl each of the diluted aqueous solution (equivalent to 100 ng of thehuman TAK1-6×His polypeptide), and the plate was incubated overnight at4° C.

[0271] After each well was washed three times with the wash buffer(diluted to 0.05% Tween 20 in PBS), 200 μl of a 5% BSA (manufactured bySIMGA) solution dissolved in PBS was added thereto and was blockedovernight at 4° C.

[0272] Then each well was washed three times with the wash buffer, 100μl of the human MBP-TAB1C-FLAG polypeptide diluted in the dilutionbuffer (1% BSA, 0.5% Tween 20, PBS) was added thereto and was incubatedat room temperature for one hour. Then each well was washed three timeswith the wash buffer, 100 μl of rabbit anti-MBP antiserum (manufacturedby New England Biolabs) diluted 5000-fold in the dilution buffer wasadded to each well and was incubated at room temperature for one hour.Then each well was washed three times with the wash buffer, 100 μl ofalkaline phosphatase-labeled goat anti-rabbit antibody (manufactured byTAGO) diluted 5000-fold in the dilution buffer was added to each welland was incubated at room temperature for one hour.

[0273] After each well was washed five times with the wash buffer, 100μl of the color development solution (the substrate buffer;p-nitrophenyl phosphate dissolved to 1 mg/ml in 50 mM NaHCO₃, 10 mMMgCl₂, pH 9.8, manufactured by Sigma) was added to each well and wasincubated at room temperature, and then absorbance at 405 nm wasdetermined using a microplate reader (Model 3550, manufactured byBIO-RAD).

[0274] The result confirmed that the absorbance increased depending onthe concentration of the recombinant MBP-TAB1C-FLAG polypeptide. On theother hand, there were no increases in absorbance dependent on theconcentration of the recombinant MBP-TA B1C-FLAG polypeptide in thegroup in which the human TAK1-6×His polypeptide was not immobilized(FIG. 2). This indicated that the contacted recombinant MBP-TAB1C-FLAGpolypeptide specifically bound to the recombinant human TAK1-6×Hispolypeptide.

[0275] (2) The purified product of human TAK1-6×His polypeptide wasdiluted in the immobilization buffer (0.1 M NaHCO₃, 0.02% NaN₃, pH 9.6).To each well of a 96-well immunoplate (manufactured by Nunc) was added100 μl each of the diluted aqueous solution (equivalent to 80 ng of thehuman TAK1-6×His polypeptide), and the plate was incubated overnight at4° C.

[0276] After each well was washed three times with the wash buffer(diluted to 0.05% Tween 20 in PBS), 200 μl of a 5% BSA.(manufactured bySIMGA) solution dissolved in PBS was added thereto and was blockedovernight at 4° C.

[0277] Then each well was washed three times with the wash buffer, and100 μl of the human TAB1-FLAG polypeptide diluted in the dilution buffer(1% BSA, 0.5% Tween 20, PBS) was added and was incubated at roomtemperature for one hour. Then each well was washed three times with thewash buffer, 100 μl of mouse anti-FLAG antibody (manufactured by IBI)diluted to 3 μg/ml in the. dilution buffer was added to each well, andwas incubated at room temperature for one hour.

[0278] Then each well was washed three times with the wash buffer, 100μl of alkaline phosphatase-labeled goat anti-mouse IgG antibody(manufactured by ZYMED) diluted 1000-fold in the dilution buffer wasadded to each well and was incubated at room temperature for one hour.After each well was washed five times with the wash buffer, 100 μl ofthe color development solution (the substrate buffer; p-nitrophenylphosphate dissolved to 1 mg/ml in 50 mM NaHCO₃, 10 mM MgCl₂, pH 9.8,manufactured by Sigma) was added to each well and was incubated at roomtemperature, and then absorbance at 405 nm was determined using amicroplate reader (Model 3550, manufactured by BIO-RAD).

[0279] The result confirmed that the absorbance increased depending onthe concentration of the recombinant human TAB1-FLAG polypeptide. On theother hand, there were no increases in absorbance dependent on theconcentration of the human TAB1-FLAG polypeptide in the group in whichthe human TAK1-6×His polypeptide was not immobilized (FIG. 3).

[0280] This indicated that the recombinant human TAK1-6×His polypeptidethat was prepared in the baculovirus expression system specificallybound to the human TAB1-FLAG polypeptide in vitro.

Example 5-2 A Binding-Inhibition Study Using the Recombinant HumanTAB1-FLAG Polypeptide

[0281] It was investigated whether the recombinant human TAB1-FLAGpolypeptide used as an inhibiting substance inhibits binding between therecombinant MBP-TAB1C-FLAG polypeptide and the recombinant TAK1polypeptide.

[0282] In a similar manner to that in the above 5-1 (2), the recombinanthuman TAK1-6×His polypeptide was immobilized and was blocked. Then therecombinant human TAB1-FLAG polypeptide that was serially diluted in thedilution buffer was added as a binding-inhibiting substance to each welltogether with 16.5 μl each of the recombinant MBP-TAB1C-FLAG polypeptideand incubated.

[0283] Thereafter, absorbance was determined as in the above, the resultof which confirmed the decrease in absorbance dependent on theconcentration of the recombinant human TAB1-FLAG polypeptide added as abinding-inhibiting substance (FIG. 4).

[0284] The foregoing has shown that the in vitro binding-evaluationsystem constructed in Example 2-1 is effective as a system for screeningsubstances that inhibit binding between the TAK1 polypeptide and theMBP-TAB1C-FLAG polypeptide.

Example 6 Construction of the TAK1-DN Expression Vector andEstablishment of the Recombinant

[0285] In order to demonstrate that the signal transduction of TGF-β canbe inhibited by inhibiting specific binding between the human TAK1polypeptide and the human TAB1 polypeptide, TAK1-DN expression vectorthat acts as a dominant negative inhibitor was constructed, wasintroduced into various cells described below, and the reactivity toTGF-β was characterized.

[0286] TAK1-DN has an amino acid sequence comprising amino acid Glu atposition 77 to amino acid Gln at position 303 of the amino acid sequenceas set forth in SEQ ID NO: 4 which is the TAB1 binding site of the TAK1polypeptide. The gene fragment encoding TAK1-DN was amplified usingph-TAK1 (Japanese Unexamined Patent Publication (Kokai) No.9(1997.)-163990) by the PCR method. Thus, using a sense primer TAK1S(SEQ ID NO: 18) containing the restriction enzyme EcoRI recognition siteand the initiation codon ATG and an antisense primer TAK1S (SEQ ID NO:19) containing the restriction enzyme NotI recognition site and the stopcodon, a DNA fragment encoding TAK1-DN was amplified.

[0287] The PCR products thus , obtained were digested with restrictionenzymes EcoRI and NotI, and then were inserted into an EcoRI recognitionsite of an animal cell expression vector pCOS1 containing EF1-α promoterand the neomycin resistant gene to produce an expression vector pTAK1DN.The expression vector pCOS1 was constructed by deleting the genecontained from plasmid HEF-PMh-gγ1 (see WO 92-19759) by digesting withEcoRI and Sam I and then ligating the EcoRI-NotI-bamHI Adaptor(manufactured by Takara Shuzo).

[0288] Then, pTAK1DN or pCOS1 that contains no inserted genes as acontrol vector was linearized by digesting with a restriction enzymePvuI. These linearized vectors were introduced by electroporation intohuman fibroblast-derived HT-1080 (ATCC CCL 121), a mouse kidneymesangial cell line SV40MES13 (ATCC CRL 1927), and a mink pulmonaryepithelial cell line Mv1Lu (ATCC CCL 64), and cells into which the genewas introduced were selected using G418 (manufactured by GIBCO-BRL ).

[0289] Expression of each gene was confirmed by the RT-PCR method usingprimer TA5 (SEQ ID NO: 20). and primer HG1-R1 (SEQ ID NO: 21). Thus,mRNA was isolated from the gene-introduced cells using the Quick PrepmRNA Micro Purification kit (manufactured by Pharmacia). Then using theFirst Strand cDNA Synthesis kit (manufactured by Pharmacia), cDNA wassynthesized from 150 ng of mRNA. The introduction of the gene wasconfirmed using 5 μl of cDNA reaction mixture as a template.

Example 7 The Action of TGF-β in Human Fibroblast-Derived HT-1080

[0290] The human fibroblast-derived HT-1080 cells (HT/DN2 and HT/DN14)into which pTAK1DN had been introduced and the control cells (HT/NEO)into which pCOS1 containing no inserted genes had been introduced wereincubated in a low-serum medium (Medium 199 containing 0.2% FBS;manufactured by GIBCO BRL) with or without 1 ng/ml TGF-β (manufacturedby King Jozo) for 24 hours. The amount of fibronectin in the culturesupernatant or the extracellular matrix extract prepared using 1 M ureasolution (1 M urea, 1 mM DTT, 10 mM Tris-HCl, pH 7.4, 10 mM EDTA,Protease inhibitor cocktail.(Complete™, manufactured by BoehringerMannheim)) was determined by the EIA method.

[0291] Thus, 100 μl of the culture supernatant or the extracellularmatrix extract prepared using 1 M urea solution was added to a 96-wellmicrotiter plate (manufactured by Nunc), and the plate was incubatedovernight at 4° C. After washing, it was blocked using a 1% BSA solution(50 mM Tris-HCl, pH 8.0, 1 mM MgCl₂, 150 mM NaCl, 0.05% Tween 20, 0.02%sodium azide), and then 10000 fold-diluted rabbit anti-human fibronectinantibody (manufactured by CALBIOCHEM) was added to the above 1% BSAsolution, and was further incubated at room temperature for 2 hours.

[0292] After washing, alkaline phosphatase-labeled goat anti-rabbit Igantibody (manufactured by TAGO) was added, and was further incubated atroom temperature for one hour. Then the substrate solution(p-nitrophenyl phosphate; manufactured by Sigma) was added andabsorbance at 450 nm was measured. As a standard, human fibronectin(manufactured by Cappel) was used.

[0293] The results are shown in FIGS. 5A and B. In FIGS. 5A and B, thenumerical values indicate the mean +/− SD of the culture supernatant andthe extracellular matrix extracts prepared using 1 M urea, each preparedfrom 3 wells.

[0294] In the control cells HT/NEO, TGF-β addition increased fibronectinin the culture supernatant by about 6.1 fold, and that in theextracellular matrix extract by about 11.4 fold. On the other hand, inHT/DN2 and HT/DN14, cells that express TAK1-DN, fibronectin in theculture supernatant increased by about 2.6 and 3.0 fold, respectively,and that in the extracellular matrix extract increased by about 3.5 and3.6 fold, respectively. These results indicated that the production offibronectin by TGF-β and the incorporation of fibronectin into thematrix were suppressed by the expression of TAK1-DN.

Example 8 Effect of TGF-β on Mouse Kidney Mesangial Cell Line SV40MES13

[0295] The mouse mesangial cell line SV40MES13 (MES/DN3 and MES/DN6)into which pTAK1DN had been introduced and the control cells (MES/NEO)into which pCOS1 containing no inserted genes had been introduced wereincubated in a low-serum medium (Medium 199 containing 0.2% FBS) with orwithout 2.5 ng/ml TGF-β for 24 hours. The amount of fibronectin in theculture supernatant or the extracellular matrix extract prepared using 1M urea solution was determined by the EIA method as described in Example7

[0296] The results are shown in FIGS. 6A and B. In FIGS. 6A and B. thenumerical values indicate the mean +/− SD of the culture supernatant andthe extracellular matrix extracts prepared using 1 M urea, each preparedfrom 3 wells. In the control cells MES/NEO, TGF-β addition increasedfibronectin in the culture supernatant by about 3.3 fold, and that inthe extracellular matrix extract by about 3.8 fold. On the other hand,in MED/DN3 and MES/DN6, cells that express TAK1-DN, fibronectin in theculture supernatant increased by about 2.4 and 2.3 fold, respectively,and that in the extracellular matrix extract increased by about 2.6 and2.1 fold, respectively. These results indicated that the production offibronectin by TGF-β and the incorporation of fibronectin into thematrix were suppressed by the expression of TAK1-DN.

[0297] Furthermore, the amount of type I and type IV collagen in theculture supernatant was determined by the EIA method. Thus, 100 μl ofthe culture supernatant was added to a 96-well microtiter plate(manufactured by Nunc), and the plate was incubated overnight at 4° C.After washing, it was blocked using the above 1% BSA solution, and then5000-fold diluted rabbit anti-mouse type I collagen antibody(manufactured by LSL) or rabbit anti-mouse type IV collagen antibody(manufactured by LSL) was added, and was further incubated at roomtemperature for 2 hours. After washing, alkaline phosphatase-labeledgoat anti-rabbit Ig antibody (manufactured by TAGO) was added, and wasfurther incubated at room temperature for one hour. Then the substratesolution (manufactured by Sigma) was added and absorbance at 450 nm wasmeasured. As a standard, mouse type I collagen (manufactured byChemicon) or mouse type IV collagen (manufactured by Cosmobio) was used.

[0298] The results are shown in FIGS. 7 and 8. In FIGS. 7 and 8, thenumerical values indicate the mean +/− SD of the culture supernatant andthe extracellular matrix extracts prepared using 1 M urea, each preparedfrom 3 wells. In the control cells MES/NEO, TGF-β addition increasedtype I collagen in the culture supernatant by about 3.6 fold, and typeIV collagen by about 2.0 fold. On the other hand, in MED/DN3 andMES4DN6, cells that express TAK1-DN, type I collagen in the culturesupernatant increased by about 2.0 and 2.0 fold, respectively, and typeIV collagen increased by about 1.5 and 1.4 fold, respectively. Theseresults indicated that the production of type I collagen and type IVcollagen by TGF-β was suppressed by the expression of TAK1-DN.

Example 9 Effect of TGF-β on Mink Epithelial Cell Line Mv1Lu

[0299] It is known that the cellular growth of mink epithelial cell lineMv1Lu is stopped at the G1 phase by stimulation with TGF-β .Accordingly, effects on the inhibition of cellular growth of TAK1-DN canbe investigated using the mink epithelial cell line Mv1Lu.

[0300] The mink epithelial cell line Mv1Lu (Mv/DN1 and Mv/DN4) intowhich pTAK1DN had been introduced and the control cells (Mv/NEO) intowhich pCOS1 containing no inserted genes had been introduced wereincubated in a low-serum medium (Medium 199 containing 0.2% FBS) with orwithout various concentrations of TGF-β for 24 hours. Then BrdU(manufactured by Boehringer Mannheim) was added to a final concentrationof 1 mM and was further incubated for 4 hours. Cellular growth could beevaluated by determining the amount of BrdU incorporated into the cellsaccording to the instructions by the manufacturer.

Example 10 Construction of TAB1 Deletion Mutant Expression Vector

[0301] In order to determine the region in the TAB1 polypeptide that isrequired for binding to the TAK1 polypeptide by the yeast 2-hybridmethod, expression vectors for TAB1 deletion mutants were constructed.Thus, it was designed that the TAB1 partial polypeptide can be expressedas a fusion polypeptide with the GAL4 transcription activated domainpolypeptide in yeast cells by constructing a gene fragment encoding apartial polypeptide of the TAB1 polypeptide, which is then inserted intoa yeast 2-hybrid expression vector pGAD10 (manufactured by CLONTECH),and was so constructed.

Example 10-1 Construction of Deletion Mutants from the Amino Terminal

[0302] TAB1C45, TAB1C25, TAB1C24, TAB1C23, TAB1C22, TAB1C21 and TAB1C20are polypeptides that comprise 45, 25, 24, 23, 22, 21 and 20 aminoacids, respectively, of the carboxy terminal of TAB1 (FIG. 11). Anexpression vector that expresses each of TAB1C45, TAB1C25, TAB1C24,TAB1C23, TAB1C22, TAB1C21 and TAB1C20 as a fusion polypeptide with theGAL4 transcription activated domain polypeptide was constructed asfollows:

[0303] The gene fragment that encodes TAB1C45 was amplified by the PCRmethod using pGAD-TAB1 (Shibuya H. et al., Science (1996) 272,1179-1182) as a template. pGAD-TAB1 is a yeast 2-hybrid expressionvector that expresses a fusion polypeptide of the 68 amino acids of thecarboxy terminal of TAB1 and the GAL4 transcription activated domainpolypeptide. Primers used are a sense primer TABC45 (SEQ ID NO: 24) thatcontains a restriction enzyme XhoI recognition sequence and an antisenseprimer TABCapEc (SEQ ID NO: 25) that contains a restriction enzyme EcoRIrecognition sequence and 2 stop codons. The PCR method comprised a totalof 15 cycles of 94° C. for 1 minute, 55° C. for 1 minute, and 72° C. for1 minute per cycle.

[0304] PCR reaction products were separated and purified by a 1%low-melting point agarose gel (manufactured by Sigma), digested withrestriction enzymes XhoI and EcoRI, and then were inserted into a yeast2-hybrid expression vector pGAD-TAB1 (manufactured by CLONTECH) toobtain a plasmid pGAD-TAB1C45 that expresses a fusion polypeptide ofTAB1C45 and the GAL4 transcription activated domain.

[0305] A gene fragment that encodes TAB1C25 was amplified using a senseprimer C25X (SEQ ID NO: 26) containing a restriction enzyme XhoIrecognition sequence and an antisense primer TABCapEc (SEQ ID NO: 25) bythe PCR method with plasmid pGAD-TAB1C45 as a template, and plasmidpGAD-TAB1C25 that expresses a fusion polypeptide of TAB1C25 and the GAL4transcription activated domain was obtained.

[0306] A gene fragment that encodes TAB1C24 was amplified using a senseprimer C24X (SEQ ID NO: 27) containing a restriction enzyme XhoIrecognition sequence and an antisense primer TABCapEc (SEQ ID NO: 25) bythe PCR method with plasmid pGAD-TAB1C45 as a template, and plasmidpGAD-TAB1C24 that expresses a fusion polypeptide of TAB1C24 and the GAL4transcription activated domain was obtained.

[0307] A gene fragment that encodes TAB1C23 was amplified using a senseprimer C23X (SEQ ID NO: 28) containing a restriction enzyme XhoIrecognition sequence and an antisense primer TABCapEc (SEQ ID NO: 25) bythe PCR method with plasmid pGAD-TAB1C45 as a template, and plasmidpGAD-TAB1C23 that expresses a fusion polypeptide of TAB1C23 and the GAL4transcription activated domain was obtained.

[0308] A gene fragment that encodes TAB1C22 was amplified using a senseprimer C22X (SEQ ID NO: 29) containing a restriction enzyme XhoIrecognition sequence and an antisense primer TABCapEc (SEQ ID NO: 25) bythe PCR method with plasmid pGAD-TAB1C45 as a template, and plasmidpGAD-TAB1C22 that expresses a fusion polypeptide of TAB1C22 and the GAL4transcription activated domain was obtained.

[0309] A gene fragment that encodes TAB1C21 was amplified using a senseprimer C21X (SEQ ID NO: 30) containing a restriction enzyme XhoIrecognition sequence and an antisense primer TABCapEc (SEQ ID NO: 25) bythe PCR method with plasmid pGAD-TAB1C45 as a template, and plasmidpGAD-TAB1C21 that expresses a fusion polypeptide of TAB1C21 and the GAL4transcription activated domain was obtained.

[0310] A gene fragment that encodes TAB1C20 was amplified using a senseprimer C20X (SEQ ID NO: 31) containing a restriction enzyme XhoIrecognition sequence and an antisense primer TABCapEc (SEQ ID NO: 25) bythe PCR method with plasmid pGAD-TAB1C45 as a template, and plasmidpGAD-TAB1C20 that expresses a fusion polypeptide of TAB1C20 and the GAL4transcription activated domain was obtained.

Example 10-2 Deletion from the Carboxy Terminal

[0311] Polypeptides that further lack polypeptides sequentially from thecarboxy terminal of the polypeptide comprising 45 amino acids of thecarboxy terminal portion of TAB1, i. e. TAB1C45 Δ14, TAB1C45 Δ19,TAB1C45 Δ20, TAB1C45 Δ21, TAB1C45 Δ22, TAB1C45 Δ23, TAB1C45 Δ24 andTAB1C45 Δ25 (FIG. 12), were designed as follows:

[0312] Thus, TAB1C45 Δ14 is a polypeptide that lacks 14 amino acids fromthe carboxy terminal of the polypeptide comprising 45 amino acids of thecarboxy terminal portion of TAB1. Similarly, TAB1C45 Δ19, TAB1C45 Δ20,TAB1C45 Δ21, TAB1C45 Δ22, TAB1C45 Δ23, TAB1C45 Δ24 and TAB1C45 Δ25 arepolypeptides that lack 19, 20, 21, 22, 23, 24 and 25 amino acids,respectively, from the carboxy terminal of the polypeptide comprising 45amino acids of the carboxy terminal portion of TAB1.

[0313] Plasmids that express these polypeptides as fusion polypeptideswith the GAL4 transcription activated domain were constructed asfollows:

[0314] The gene fragment that encodes TAB1C45 Δ14 polypeptide wasamplified by the PCR method using pGAD-TAB1C45 as a template. Thus, PCRwas carried out using a sense primer TABC45 (SEQ ID NO: 24) thatcontains a restriction enzyme XhoI recognition sequence and an antisenseprimer TABCD14A (SEQ ID NO: 32) that contains a restriction enzyme EcoRIrecognition sequence and 2 stop codons. The PCR method comprised a totalof 15 cycles of 94° C. for 1 minute, 55° C. for 1 minute, and 72° C. for1 minute per cycle.

[0315] PCR reaction products were separated and purified by a 1%low-melting point agarose gel (manufactured by Sigma), digested withrestriction enzymes XhoI and EcoRI, and then were inserted into a yeast2-hybrid expression vector pGAD10 (manufactured by CLONTECH) to obtain aplasmid pGAD-TAB1C45D14 that expresses a fusion polypeptide of TAB1C45Δ14 polypeptide and the GAL4 transcription activated domain.

[0316] A gene fragment that encodes TAB1C45 Δ19 was amplified using asense primer TABC45 (SEQ ID NO: 24) and an antisense primer TABCD19A(SEQ ID NO: 33) containing a restriction enzyme EcoRI recognitionsequence and 2 stop codons by the PCR method with plasmid pGAD-TAB1C45as a template, and thereby plasmid pGAD-TAB1C45D19 that expresses afusion polypeptide of TAB1C45 Δ19 and the GAL4 transcription activateddomain was obtained.

[0317] A gene fragment that encodes TAB1C45 Δ20 was amplified using asense primer TABC45 (SEQ ID NO: 24) and an antisense primer TABCD20 (SEQID NO: 34) containing a restriction enzyme EcoRI recognition sequenceand 2 stop codons by the PCR method with plasmid pGAD-TAB1C45 as atemplate, and thereby plasmid pGAD-TAB1C45D20 that expresses a fusionpolypeptide of TAB1C45 Δ20 and the GAL4 transcription activated domainwas obtained.

[0318] A gene fragment that encodes TAB1C45 Δ21 was amplified using asense primer TABC45 (SEQ ID NO: 24) and an antisense primer TABCD21 (SEQID NO: 35) containing a restriction enzyme EcoRI recognition sequenceand 2 stop codons by the PCR method with plasmid pGAD-TAB1C45 as atemplate, and thereby plasmid pGAD-TAB1C45D21 that expresses a fusionpolypeptide of TAB1C45 Δ21 and the GAL4 transcription activated domainwas obtained.

[0319] A gene fragment that encodes TAB1C45 Δ22 was amplified using asense primer TABC45 (SEQ ID NO: 24) and an antisense primer TABCD22 (SEQID NO: 36) containing a restriction enzyme EcoRI recognition sequenceand 2 stop codons by the PCR method with plasmid pGAD-TAB1C45 as atemplate, and thereby plasmid pGAD-TAB1C45D22 that expresses a fusionpolypeptide of TAB1C45 Δ22 and the GAL4 transcription activated domainwas obtained.

[0320] A gene fragment that encodes TAB1C45 Δ23 was amplified using asense primer. TABC45 (SEQ ID NO: 24) and an antisense primer TABCD23(SEQ ID NO: 37) containing a restriction enzyme EcoRI recognitionsequence and 2 stop codons by the PCR method with plasmid pGAD-TAB1C45as a template, and thereby plasmid pGAD-TAB1C45D23 that expresses afusion polypeptide of TAB1C45 Δ23 and the GAL4 transcription activateddomain was obtained.

[0321] A gene fragment that encodes TAB1C45 Δ24 was amplified using asense primer TABC45 (SEQ ID NO: 24) and an antisense primer TABCD24 (SEQID NO: 38) containing a restriction enzyme EcoRI recognition sequenceand 2 stop codons by the PCR method with plasmid pGAD-TAB1C45 as atemplate, and thereby plasmid pGAD-TAB1C45D24 that expresses a fusionpolypeptide of TAB1C45 Δ24 and the GAL4 transcription activated domainwas obtained.

[0322] A gene fragment that encodes TAB1C45 Δ25 was amplified using asense primer TABC45. (SEQ ID NO: 24) and an antisense primer TABCD25(SEQ ID NO: 39) containing a restriction enzyme EcoRI recognitionsequence and 2 stop codons by the PCR method with plasmid pGAD-TAB1C45as a template, and thereby plasmid pGAD-TAB1C45D25 that expresses afusion polypeptide of TAB1C45 Δ25 and the , GAL4 transcription activateddomain was obtained.

Example 11 Transformation of Yeast

[0323] In order to evaluate each TAB1 deletion mutant constructed inExample 10, a yeast 2-hybrid expression vector of each TAB1 deletionmutant and a yeast 2-hybrid expression vector pBTMHu11F (Shibuya H. etal., Science (1996) 272, 1179-1182) that expresses TAK1 wereco-transformed into a yeast strain L40 (Shibuya H. et al., Science(1996) 272, 1179-1182). One mg each of a TAB1 deletion mutant expressionvector (pGAD-TAB1C45 to pGAD-TAB1C45 in Working Example 10-1 andpGAD-TAB1C45D14 to pGAD-TAB1C45D25 in Working Example 10-2) or pGAD-TAB1(Shibuya H. et al., Science (1996) 272, 1179-1182) as a control andpBTMHu11F were introduced into the L40 strain according to theinstructions (MATCHMAKER™ Two-Hybrid System, manufactured by CLONTECH),and were incubated on a selection agar medium SD-ULW (glucose 20 g, agar(manufactured by DIFCO) 20 g, Yeast Nitrogen Base w/o amino acids(manufactured by DIFCO) 6.7 g, adenine 100 mg, isoleucine 30 mg, valine150 mg, arginine 20 mg, lysine 30 mg, methionine 20 mg, phenylalanine 50mg, threonine 200 mg, tyrosine 30 mg, histidine 100 mg per liter ofmedium) at 30° C. for 3 days to obtain each transformant.

Example 12 Evaluation of the Binding Ability of TAB1 Deletion Mutants toTAK1

[0324] In order to evaluate the binding ability to TAK1 of each TAB1deletion mutant constructed in Example 10, activity was determined bythe yeast 2-hybrid method. Since a reporter gene lacZ having the LexAbinding sequence upstream thereof has been integrated on the chromosomeof the yeast strain L40, the binding ability to TAK1 of each TAB1deletion mutant can be evaluated in relative terms by measuring theactivity of β-galactosidase that is a reporter gene product.

Example 12-1 Evaluation of Deletion Mutants (TAB1C45 to TAB1C20) fromthe Amino Terminal

[0325] The β-galactosidase activity of each transformant obtained inExample 11 was determined according to the instructions (MATCHMAKER™Two-Hybrid System, manufactured by CLONTECH), and the activity wascalculated using Miller Unit (Miller, J. H. (1972) Experiments inMolecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.).

[0326] The results are shown in FIG. 11. In FIG. 11, the β-galactosidaseactivity of each TAB1 deletion mutant and the yeast L40 transformed bythe yeast 2-hybrid expression plasmid of TAK1 was expressed in terms ofMiller Units. Measurement was carried out three times and the mean +/−SD is shown. The value indicates a ratio based on the β-galactosidaseactivity of the yeast L40 transformed by TAB1C68 and the yeast 2-hybridexpression plasmid of TAK1

[0327] Since specific activities of TAB1C25 and TAB1C24 are 0.28 and0.35, respectively, whereas those of TAB1C23, TAB1C22, TAB1C21 andTAB1C20 markedly decrease to 0.05, 0.03, 0.03 and 0.03, respectively,the amino terminal of the region required for binding of TAB1 to TAK1 isbelieved to be the amino terminal Tyr residue (amino acid position 481in the amino acid sequence as set forth in SEQ ID NO: 2) of TAB1C24.

Example 12-2 Evaluation of Deletion Mutants (TAB1C45 Δ14 to TAB1C45 Δ25)from the Carboxy Terminal

[0328] The β-galactosidase activity of each transformant obtained inExample 11 was determined according to the instructions (MATCHMAKER™Two-Hybrid System, manufactured by CLONTECH), and the activity wascalculated using Miller Units (Miller, J. H. (1972) Experiments inMolecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.).

[0329] The results are shown in FIG. 12. In FIG. 12, the β-galactosidaseactivity of the yeast L40 transformed by each TAB1 deletion mutant andthe yeast 2-hybrid expression plasmid of TAK1 was expressed in terms ofMiller Units. Measurement was carried out three times and the mean +/−SD is shown. The value indicates a ratio based on the β-galactosidaseactivity of the yeast L40 transformed by the TAB1C68 and yeast 2-hybridexpression plasmid of TAK1.

[0330] Since specific activities of TAB1C45 Δ19 and TAB1C45 Δ20are 0.13and 0.11, respectively, whereas those of TAB1C45 Δ21, TAB1C45 Δ22,TAB1C45 Δ23, TAB1C45 Δ24 and TAB1C45 Δ25 markedly decrease to 0.01,0.02, 0.00, 0.02 and 0.01, respectively, the carboxy terminal of theregion required for binding of TAB1 to TAK1 is believed to be thecarboxy terminal Phe residue (amino acid position 484 in the amino acidsequence as set forth in SEQ ID NO: 2) of TAB1C45 Δ20.

[0331] From the foregoing, the region required for binding of TAB1 toTAK1 is believed to be the region from Tyr at amino acid position 481 toPhe at amino acid position 484 of the amino acid sequence as set forthin SEQ ID NO: 2.

Example 13 Binding-Inhibition Study Using Synthetic Peptides

[0332] The fact that a synthetic peptide containing the amino acidsequence identified as the TAK1 binding region can inhibit bindingbetween TAK1 and TAB1 was confirmed by the following experiment. Thus, apeptide TAB1C-1 (SEQ ID NO: 40) comprising 16 amino acid residuescontaining the TAK1 binding region of TAB1 described in the aboveExample and a control peptide TAB1C-2 (SEQ ID NO: 41) containing anamino acid sequence from Gln at position 437 to Gln at position 451 ofTAB1 were each synthesized, and were evaluated for their effects on thebinding between TAK1 and TAB1. The TAK1 and TAB1 used in the presentinvention were prepared in the following manner. Thus, the TAK1expression vector or the TAB1 expression vector, was introduced intoCOS-7 cells using LIPOFECTOAMINE (manufactured by GIBCO-BRL) by astandard method. After incubating for 72 hours, the cells were harvestedand washed with PBS. Subsequently, they were suspended in the lysisbuffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40,Complete Protease Inhibitor Cocktail (Boehringer Mannheim)), incubatedat 4° C. for 1 hour, and the insoluble components were removed bycentrifugation to prepare each cell extract.

[0333] The TAK1 expression vector pCOS-TAK1 was constructed as describedbelow. Thus, plasmid pBacTABF having a nucleotide sequence encodingTAK1-6×His was digested with restriction enzymes EcoRI and NotI, and agene fragment containing a nucleotide sequence encoding TAK1-6×His waspurified using a 1.5% low-melting point agarose gel (manufactured bySigma) and was inserted to an expression vector pCOS1 to constructpCOS-TAK1. The TAB1 expression vector pCOS-FTAKB1 was constructed asdescribed below. Thus, a gene fragment encoding RLAG-TAB1 (SEQ ID NO:42) to which was added a FLAG tag comprising 8 amino acids to the aminoterminal of TAB1 was amplified by the PCR method using pBacTABF as atemplate DNA. The PCR method was carried out as described above usingEco-MetFTAB (SEQ ID NO: 44) as a sense primer that was designed tocontain a nucleotide sequence encoding a restriction enzyme EcoRIrecognition site, ATG initiation codon, and a FLAG tag and TABC-Not (SEQID NO: 45) as an antisense primer that was designed to contain a stopcodon and a restriction enzyme NotI recognition site. The PCR productwas digested with restriction enzymes EcoRI and NotI, inserted intopCOS1, and the plasmid that has the correct base sequence was used asthe expression vector pCOS-FTAB1.

[0334] Subsequently, cell extracts each containing TAK1-6×His orFLAG-TAB1 and a 5 mM synthetic peptide were mixed to a finalconcentration of 50 or 500 mM, respectively, and then incubatedovernight at 4° C. It was then immunoprecipitated using anti-TAK1antibody, and the amount of bound TAK1 and TAB1 was evaluated bydetermining the amount of TAB1 that coprecipitated by Western analysis.Thus, 2 mg each of anti-TAK1 polyclonal antibody (manufactured bySantaCruz) was added to each reaction mixture, and further incubated at4° C. for 1 hour. Then 40 μl (50% v/v) of Protein-G Sepharose(manufactured by Pharmacia) was added thereto, and was further incubatedfor 1 hour. After the immunoprecipitate was washed three times with TBS.containing 0.05% Tween 20, it was subjected to SDS-PAGE. Theimmunoprecipitate separated by SDS-PAGE was transferred to anitrocellulose membrane (manufactured by Schleicher & Schuell) and wassubjected to a Western analysis. After it was blocked using a TBSsolution containing 5% BSA, TAK1 in the immunoprecipitate was detectedusing anti-TAK1 antibody (manufactured by SantaCruz) and thecoprecipitataed FLAG-TAB1 was detected using anti-FLAG M2 antibody(manufactured by Kodak). The amount of TAK1 and FLAG-TAB1 was eachdetermined by quantifying each band using an analysis software QuantityOne (manufactured by PDI), and the amount of the coprecipitatedFLAG-TAB1 was corrected with the amount of TAK1 to obtain the amountbound of TAK1 and TAB1.

[0335] The result is shown in FIG. 13. Compared to the , case wherein nosynthetic peptides were added, no decrease in binding between TAK1 andTAB1 was observed when the control peptide TAB1C-2 was added, whereas adecrease in the amount of coprecipitated FLAG-TAB1 was observed whenTAB1C-1 was added. The above result indicates that a synthetic peptidecontaining the TAK1 binding region has an activity of inhibiting bindingbetween TAK1 and TAB1. Accordingly, it is believed that syntheticpeptides containing the amino acid sequence , identified as the TAK1binding region or derivatives thereof and substances that act on thatregion act on binding between TAK1 and TAB1 and activate or inhibitsignal transduction from TAK1.

Example 14 Identification of the TAB1 Region Essential for the Inductionof TAK1 Activation

[0336] The region of TAB1 required to induce the kinase activity afterbinding to TAK1 was identified using TAB1 deletion mutants.

[0337] Expression vectors for the 40, 35 and 30 amino acid regions (FIG.14) in the carboxy terminal of TAB1 were constructed as described below.Thus ,, genes encoding 40, 35 and 30 amino acid regions respectively inthe carboxy terminal of TAB1 were amplified by the PCR method asdescribed above, digested with restriction enzymes XhoI and EcoRI, andinserted to the GAL4 transcription activated domain expression vectorpGAD10 to construct pGAD-TAB1C40, pGAD-TAB1C35 and pGAD-TAB1C30,respectively. Furthermore, expression vectors for the TAB1 deletionmutants (FIG. 14) comprising 68, 45 and 25 amino acid regions in thecarboxy terminal of TAB1 used were pGAD-TAB1, pGAD-TAB1C45 andpGAD-TAB1C25.

[0338] The gene encoding the 40 amino acid region of the carboxyterminal of TAB1 was amplified using a sense primer TABC40 (SEQ ID NO:46) containing a restriction enzyme XhoI recognition sequence and anantisense primer TABCapEc (SEQ ID NO: 25) by the PCR method with plasmidpGAD-TAB1C45 as a template DNA.

[0339] The gene encoding the 35 amino acid region of the carboxyterminal of TAB1 was amplified using a sense primer TABC35 (SEQ ID NO:47) containing a restriction enzyme XhoI recognition sequence and anantisense primer TABCapEc (SEQ ID NO: 25) by the PCR method with plasmidpGAD-TAB1C45 as a template DNA.

[0340] The gene encoding the 30 amino acid region of the carboxyterminal of TAB1 was amplified using a sense primer TABC30 (SEQ ID NO:48) containing a restriction enzyme XhoI recognition sequence and anantisense primer TABCapEc (SEQ ID NO: 25) by the PCR method with plasmidpGAD-TAB1C45 as a template DNA.

[0341] First, binding between the 68, 45, 40, 35, 30 and 25 amino acidregions of the carboxy terminal of TAB1 and TAK1 was evaluated by theyeast 2-hybrid method mentioned above.

[0342] Subsequently, it was investigated whether each TAB1 deletionmutant can induce the kinase activity of TAK1 using the method describedin Japanese Unexamined Patent Publication (Kokai) 9(1997)-163990. Thus,the above TAB1 deletion mutant expression vector and TAK1 expressionvector pNVll-HUll (Yamaguchi, K. et al., Science (1995) 270, 2008-2011)were introduced into Saccharomyces cereviceae SY1984-P strain (his3Δ,stellΔ, FUS1p: :HIS3, STE7^(P368)) to obtain deletion mutants. In thisyeast strain, the original his3 is lacking, and hence it can only growwhen foreign histidine is present in the medium or when the lackingStell activity is complemented by mutation. These transformants wereplated onto a SC-His (glucose 20 g, agar (manufactured by DIFCO) 20 g,Yeast Nitrogen Base w/o amino acids (manufactured by DIFCO) 6.7 g,adenine 100 mg, isoleucine 30 mg, valine 150 mg, arginine 20 mg, lysine30 mg, methionine 20 mg, phenylalanine 50 mg, threonine 200 mg, tyrosine30 mg per liter of medium) plate containing no histidine, incubated at30° C., and the growth of yeast transformed with each expression vectorwas confirmed in order to evaluate the ability of each TAB1 deletionmutant to activate TAK1.

[0343] These results are shown in FIG. 14. Binding to TAK1 was observedfor all TAB1 deletion mutants whereas the ability to activate TAK1 foreach TAB1 mutant was observed for TAB1C68, 45, 40, 35 and 30 but not forTAB1C25. From these results, it is believed that the region importantfor TAK1 activation is present in between No. 30 and No. 26(corresponding to Asp at position 475 to Glu at position 479 in theamino acid sequence of SEQ ID NO: 2) from the carboxy terminal. Thus,peptides that lack an amino acid sequence of this region, or peptidesthat contain this amino acid sequence or derivatives thereof as well assubstances that act on this region can serve as an inhibitor or induceror a stimulator of TAK1 activation.

Reference Example 1

[0344] It was analyzed whether a polypeptide (TAK1-DN) comprising Glu atposition 77 to Gln at position 303 of the TAK1 polypeptide as set forthin SEQ ID NO: 4 inhibits binding between the TAK1 polypeptide and theTAB1 polypeptide and whether it can inhibit, the activation of the TAK1polypeptide in animal cells by an animal cell 2-hybrid system (Dang etal., (1991) Mol. Cell. Biol. 11, 954-962) using the TAB1 polypeptide andthe TAK1 polypeptide.

[0345] First, a gene encoding a full-length TAK1 and TAK1-DN and a geneencoding the GAL4 DNA-binding domain (GAL4-BD) were ligated to constructan expression vector. A gene encoding a full-length TAK1 was prepared bydigesting yeast 2-hybrid expression plasmid pBTMHu11F (Shibuya H. etal., (1996) 272, 1179-1182) with restriction enzymes EcoRI and PstI, andwas then linked to the EcoRI/Pst site of an expression vector pM(manufactured by CLONTECH) containing the GAL4-BD gene, which was termedan animal cell 2-hybrid expression plasmid pM-TAK1.

[0346] Subsequently, a gene encoding TAK1-DN was amplified using a senseprimer DNTAK5′ (SEQ ID NO: 22) to which a restriction enzyme EcoRIrecognition site had been added and an antisense primer DNTAK3′ (SEQ IDNO: 23) to which a restriction enzyme PstI recognition site had beenadded by PCR with plasmid pBTMHu11F as a template DNA. After digestionwith restriction enzymes EcoRI and PstI, it was ligated to the pM vectorto obtain an animal cell 2-hybrid expression plasmid pM-TAKlDN.

[0347] Then, a gene encoding TAB1C68 comprising 68 amino acid residuesof the carboxy terminal of the TAB1 polypeptide and a gene encoding VP16protein-derived transcription activated domain (VP16-AD) of herpessimplex virus were ligated to construct an expression vector. A geneencoding TAB1C68 was prepared by digesting yeast 2-hybrid expressionplasmid pGAD-TAB1 (Shibuya H. et al., (1996) 272, 1179-1182) with arestriction enzyme EcoRI, and was then linked to the EcoRI site of anexpression vector pVP16 (manufactured by CLONTECH) containing a geneencoding VP16-AD, which was termed an animal cell 2-hybrid expressionplasmid pVP16-C68.

[0348] The reporter plasmid used was pG-Luc in which a gene encoding CATof pG5CAT (manufactured by CLONTECH) having five contiguous GAL4 bindingsites and the chloramphenicol asetyltransferase (CAT) gene downstreamthereof replaced with the luciferase gene.

[0349] After incubating overnight CHO cells (5×10⁴ cells/well), theywere washed with PBS. Then a mixture comprising 500 ng of a GAL4-BDfusion protein expression plasmid (either of pM, pM-TAK1, andpM-TAK1DN), 500 ng of a VP16-AD fusion protein expression plasmid(either of pVp16 and pVp16-C68), 100 ng of the reporter plasmid pG5-Lucand 50 ng of pRL-SV40 (containing the luciferase gene of Renilladownstream of SV40 promoter: manufactured by Promega) and 10 ml ofLIPOFECTOAMINE (manufactured by GIBCO-BRL) was added thereto and wasincubated for 5 hours to introduce genes.

[0350] After further incubating for 72 hours, luciferase activity ineach cell extract was determined using the Dual-LuciferaseTM AssaySystem (manufactured by Promega). Thus, after the cells were washed withPBS, 250 ml of the Passive Lysis Buffer was added thereto, incubated atroom temperature for 15 minutes , and 20 μl of each was used as the cellextract for the assay. The efficiency of gene introduction wascorrected. with the measured value of luciferase activity of Renilla bypRL-SV40. The result is shown in FIG. 9.

[0351] Similarly to the combination of pM-TAK1 and pVP16-C68, increasesin luciferase activity was confirmed for the combination of pM-TAK1DNand pVP16-C68, revealing that TAK1DN, as the full-length TAK1, binds toTAB1.

Reference Example 2

[0352] Since TAK1DN does not contain lysine at position 63 of SEQ ID NO:2 that is an amino acid residue essential for ATP binding for the TAK1polypeptide to exhibit kinase activity, it is expected to exhibit nokinase activity by itself. It is also expected to inhibit the activationof endogenous TAK1 polypeptide through inhibition of binding between thefull-length TAK1 polypeptide and the TAB1 polypeptide by binding to theTAB1 polypeptide in the cell.

[0353] It has been demonstrated that PAI-1 (plasminogen activatorinhibitor type 1) expression is in creased due to stimulation by TGF-βin the Mv1Lu cell and that PAI-1 expression is inhibited by forcedexpression of a catalytically inactive TAK1 polypeptide mutant TAK1-K63W(Yamaguchi K. et al., (1995) Science 270, 2008-2011).

[0354] Accordingly, TAK1DN was subjected to forced expression in theMv1Lu cell to investigate the effects of TGF-β stimulation on PAI-1expression. The TAK1DN expression vector used was the above-mentionedTAK1DN, and the TAK1 polypeptide mutant TAK1-K63W expression vector wasconstructed by inserting a gene (Yamaguchi K. et al., (1995) Science270, 2008-2011) encoding the TAK1 polypeptide mutant TAK1-K63W in whichthe lysine residue at position 63 has been replaced with the tryptophanresidue at the EcoRI and NotI restriction enzyme site of pCOSl to givepTAK1K63W.

[0355] The Mv1Lu cells into which pTAK1Dn had been introduced (Mv/DN2),the cells into which pTAK1K63W had been introduced (Mv/KN7), and thecontrol cells into which pCOS1 containing no inserted genes (Mv/NEO) hadbeen introduced were each incubated in a low-serum medium (MEM mediumcontaining 0.2% FBS; manufactured by GIBCO-BRL) with or without 10 ng/mlof TGF-β1 for 24 hours. The amount of PAI-1 in the culture supernatantwas determined using the PAI-1 Quantative ELISA (manufactured byCALBIOCHEM). The results are shown in FIG. 10.

[0356] In the control cells there was an about 16-fold increase in PAI-1by TGF-β1 addition in the culture supernatant, whereas in the MV/KN6cells that express the TAK1 polypeptide mutant TAK1-K63W an increase inPAI-1 was about 6.5 fold and in the Mv/DN2 cells the increase was up toabout 4.3 fold. Thus, TAK1DN inhibited the effect of enhancingexpression of PAI-1 by TGF-β1 stimulation in a similar manner to theTAK1 polypeptide mutant TAK1-K63W.

[0357] The forgoing has shown that TAK1DN inhibits signal transductionvia the TAK1 polypeptide and the TAB1 polypeptide by TGF-β1 stimulation,by inhibiting binding between the endogenous TAK1 polypeptide and theTAB1 polypeptide.

[0358] Industrial Applicability

[0359] It was revealed that substances that inhibit binding between theTAB1 polypeptide and the TAK1 polypeptide can be screened by thescreening method of the present invention. The screening method of thepresent invention is useful for screening substances that inhibitbinding between the TAB1 polypeptide and the TAK1 polypeptide.Substances obtained by the screening method of the present inventionthat inhibit binding between the TAB1 polypeptide and the TAK1polypeptide are useful as pharmaceutical agents.

[0360] Reference to the microorganisms deposited under the PatentCooperation Treaty, Rule 13-2, and the name of the Depository Institute

[0361] Depository Institute

[0362] Name: the National Institute of Bioscience and Human Technology,Agency of Industrial Science and Technology Address: 1-3, Higashi1-chome, Tsukuba-shi, Ibaraki, Japan

[0363] Organism (1)

[0364] Indication: Escherichia coli MC1061/P3 (pEF-TAK1DN)

[0365] Accession number: FERM BP-5245

[0366] Date deposited: Sep. 28, 1995

[0367] Organism (2)

[0368] Indication: Escherichia coli MC1061/P3 (pEF-TAK1)

[0369] Accession number: FERM BP-5246

[0370] Date deposited: Sep. 28, 1995

[0371] Organism (3)

[0372] Indication: Escherichia coli HB101 (pBS-TAB1)

[0373] Accession number: FERM BP-5508

[0374] Date deposited: Apr. 19, 1996

[0375] Organism (4)

[0376] Indication: Escherichia coli JM109 (phTAK1)

[0377] Accession number: FERM BP-5598

[0378] Date deposited: Jul. 19, 1996

[0379] Organism (5)

[0380] Indication: Escherichia coli DH5a (TAB1-f-4)

[0381] Accession number: FERM BP-5599

[0382] Date deposited: Jul. 19, 1996

1 48 1 1560 DNA Homo sapiens CDS (30)..(1541) 1 gaattcgtgg cccgcagggttcctccaag atg gcg gcg cag agg agg agc ttg 53 Met Ala Ala Gln Arg Arg SerLeu 1 5 ctg cag agt gag cag cag cca agc tgg aca gat gac ctg cct ctc tgc101 Leu Gln Ser Glu Gln Gln Pro Ser Trp Thr Asp Asp Leu Pro Leu Cys 1015 20 cac ctc tct ggg gtt ggc tca gcc tcc aac cgc agc tac tct gct gat149 His Leu Ser Gly Val Gly Ser Ala Ser Asn Arg Ser Tyr Ser Ala Asp 2530 35 40 ggc aag ggc act gag agc cac ccg cca gag gac agc tgg ctc aag ttc197 Gly Lys Gly Thr Glu Ser His Pro Pro Glu Asp Ser Trp Leu Lys Phe 4550 55 agg agt gag aac aac tgc ttc ctg tat ggg gtc ttc aac ggc tat gat245 Arg Ser Glu Asn Asn Cys Phe Leu Tyr Gly Val Phe Asn Gly Tyr Asp 6065 70 ggc aac cga gtg acc aac ttc gtg gcc cag cgg ctg tcc gca gag ctc293 Gly Asn Arg Val Thr Asn Phe Val Ala Gln Arg Leu Ser Ala Glu Leu 7580 85 ctg ctg ggc cag ctg aat gcc gag cac gcc gag gcc gat gtg cgg cgt341 Leu Leu Gly Gln Leu Asn Ala Glu His Ala Glu Ala Asp Val Arg Arg 9095 100 gtg ctg ctg cag gcc ttc gat gtg gtg gag agg agc ttc ctg gag tcc389 Val Leu Leu Gln Ala Phe Asp Val Val Glu Arg Ser Phe Leu Glu Ser 105110 115 120 att gac gac gcc ttg gct gag aag gca agc ctc cag tcg caa ttgcca 437 Ile Asp Asp Ala Leu Ala Glu Lys Ala Ser Leu Gln Ser Gln Leu Pro125 130 135 gag gga gtc cct cag cac cag ctg cct cct cag tat cag aag atcctt 485 Glu Gly Val Pro Gln His Gln Leu Pro Pro Gln Tyr Gln Lys Ile Leu140 145 150 gag aga ctc aag acg tta gag agg gaa att tcg gga ggg gcc atggcc 533 Glu Arg Leu Lys Thr Leu Glu Arg Glu Ile Ser Gly Gly Ala Met Ala155 160 165 gtt gtg gcg gtc ctt ctc aac aac aag ctc tac gtc gcc aat gtcggt 581 Val Val Ala Val Leu Leu Asn Asn Lys Leu Tyr Val Ala Asn Val Gly170 175 180 aca aac cgt gca ctt tta tgc aaa tcg aca gtg gat ggg ttg caggtg 629 Thr Asn Arg Ala Leu Leu Cys Lys Ser Thr Val Asp Gly Leu Gln Val185 190 195 200 aca cag ctg aac gtg gac cac acc aca gag aac gag gat gagctc ttc 677 Thr Gln Leu Asn Val Asp His Thr Thr Glu Asn Glu Asp Glu LeuPhe 205 210 215 cgt ctt tcg cag ctg ggc ttg gat gct gga aag atc aag caggtg ggg 725 Arg Leu Ser Gln Leu Gly Leu Asp Ala Gly Lys Ile Lys Gln ValGly 220 225 230 atc atc tgt ggg cag gag agc acc cgg cgg atc ggg gat tacaag gtt 773 Ile Ile Cys Gly Gln Glu Ser Thr Arg Arg Ile Gly Asp Tyr LysVal 235 240 245 aaa tat ggc tac acg gac att gac ctt ctc agc gct gcc aagtcc aaa 821 Lys Tyr Gly Tyr Thr Asp Ile Asp Leu Leu Ser Ala Ala Lys SerLys 250 255 260 cca atc atc gca gag cca gaa atc cat ggg gca cag ccg ctggat ggg 869 Pro Ile Ile Ala Glu Pro Glu Ile His Gly Ala Gln Pro Leu AspGly 265 270 275 280 gtg acg ggc ttc ttg gtg ctg atg tcg gag ggg ttg tacaag gcc cta 917 Val Thr Gly Phe Leu Val Leu Met Ser Glu Gly Leu Tyr LysAla Leu 285 290 295 gag gca gcc cat ggg cct ggg cag gcc aac cag gag attgct gcg atg 965 Glu Ala Ala His Gly Pro Gly Gln Ala Asn Gln Glu Ile AlaAla Met 300 305 310 att gac act gag ttt gcc aag cag acc tcc ctg gac gcagtg gcc cag 1013 Ile Asp Thr Glu Phe Ala Lys Gln Thr Ser Leu Asp Ala ValAla Gln 315 320 325 gcc gtc gtg gac cgg gtg aag cgc atc cac agc gac accttc gcc agt 1061 Ala Val Val Asp Arg Val Lys Arg Ile His Ser Asp Thr PheAla Ser 330 335 340 ggt ggg gag cgt gcc agg ttc tgc ccc cgg cac gag gacatg acc ctg 1109 Gly Gly Glu Arg Ala Arg Phe Cys Pro Arg His Glu Asp MetThr Leu 345 350 355 360 cta gtg agg aac ttt ggc tac ccg ctg ggc gaa atgagc cag ccc aca 1157 Leu Val Arg Asn Phe Gly Tyr Pro Leu Gly Glu Met SerGln Pro Thr 365 370 375 ccg agc cca gcc cca gct gca gga gga cga gtg taccct gtg tct gtg 1205 Pro Ser Pro Ala Pro Ala Ala Gly Gly Arg Val Tyr ProVal Ser Val 380 385 390 cca tac tcc agc gcc cag agc acc agc aag acc agcgtg acc ctc tcc 1253 Pro Tyr Ser Ser Ala Gln Ser Thr Ser Lys Thr Ser ValThr Leu Ser 395 400 405 ctt gtc atg ccc tcc cag ggc cag atg gtc aac ggggct cac agt gct 1301 Leu Val Met Pro Ser Gln Gly Gln Met Val Asn Gly AlaHis Ser Ala 410 415 420 tcc acc ctg gac gaa gcc acc ccc acc ctc acc aaccaa agc ccg acc 1349 Ser Thr Leu Asp Glu Ala Thr Pro Thr Leu Thr Asn GlnSer Pro Thr 425 430 435 440 tta acc ctg cag tcc acc aac acg cac acg cagagc agc agc tcc agc 1397 Leu Thr Leu Gln Ser Thr Asn Thr His Thr Gln SerSer Ser Ser Ser 445 450 455 tct gac gga ggc ctc ttc cgc tcc cgg ccc gcccac tcg ctc ccg cct 1445 Ser Asp Gly Gly Leu Phe Arg Ser Arg Pro Ala HisSer Leu Pro Pro 460 465 470 ggc gag gac ggt cgt gtt gag ccc tat gtg gacttt gct gag ttt tac 1493 Gly Glu Asp Gly Arg Val Glu Pro Tyr Val Asp PheAla Glu Phe Tyr 475 480 485 cgc ctc tgg agc gtg gac cat ggc gag cag agcgtg gtg aca gca ccg 1541 Arg Leu Trp Ser Val Asp His Gly Glu Gln Ser ValVal Thr Ala Pro 490 495 500 tagggcagcc ggaggaatg 1560 2 504 PRT Homosapiens 2 Met Ala Ala Gln Arg Arg Ser Leu Leu Gln Ser Glu Gln Gln ProSer 1 5 10 15 Trp Thr Asp Asp Leu Pro Leu Cys His Leu Ser Gly Val GlySer Ala 20 25 30 Ser Asn Arg Ser Tyr Ser Ala Asp Gly Lys Gly Thr Glu SerHis Pro 35 40 45 Pro Glu Asp Ser Trp Leu Lys Phe Arg Ser Glu Asn Asn CysPhe Leu 50 55 60 Tyr Gly Val Phe Asn Gly Tyr Asp Gly Asn Arg Val Thr AsnPhe Val 65 70 75 80 Ala Gln Arg Leu Ser Ala Glu Leu Leu Leu Gly Gln LeuAsn Ala Glu 85 90 95 His Ala Glu Ala Asp Val Arg Arg Val Leu Leu Gln AlaPhe Asp Val 100 105 110 Val Glu Arg Ser Phe Leu Glu Ser Ile Asp Asp AlaLeu Ala Glu Lys 115 120 125 Ala Ser Leu Gln Ser Gln Leu Pro Glu Gly ValPro Gln His Gln Leu 130 135 140 Pro Pro Gln Tyr Gln Lys Ile Leu Glu ArgLeu Lys Thr Leu Glu Arg 145 150 155 160 Glu Ile Ser Gly Gly Ala Met AlaVal Val Ala Val Leu Leu Asn Asn 165 170 175 Lys Leu Tyr Val Ala Asn ValGly Thr Asn Arg Ala Leu Leu Cys Lys 180 185 190 Ser Thr Val Asp Gly LeuGln Val Thr Gln Leu Asn Val Asp His Thr 195 200 205 Thr Glu Asn Glu AspGlu Leu Phe Arg Leu Ser Gln Leu Gly Leu Asp 210 215 220 Ala Gly Lys IleLys Gln Val Gly Ile Ile Cys Gly Gln Glu Ser Thr 225 230 235 240 Arg ArgIle Gly Asp Tyr Lys Val Lys Tyr Gly Tyr Thr Asp Ile Asp 245 250 255 LeuLeu Ser Ala Ala Lys Ser Lys Pro Ile Ile Ala Glu Pro Glu Ile 260 265 270His Gly Ala Gln Pro Leu Asp Gly Val Thr Gly Phe Leu Val Leu Met 275 280285 Ser Glu Gly Leu Tyr Lys Ala Leu Glu Ala Ala His Gly Pro Gly Gln 290295 300 Ala Asn Gln Glu Ile Ala Ala Met Ile Asp Thr Glu Phe Ala Lys Gln305 310 315 320 Thr Ser Leu Asp Ala Val Ala Gln Ala Val Val Asp Arg ValLys Arg 325 330 335 Ile His Ser Asp Thr Phe Ala Ser Gly Gly Glu Arg AlaArg Phe Cys 340 345 350 Pro Arg His Glu Asp Met Thr Leu Leu Val Arg AsnPhe Gly Tyr Pro 355 360 365 Leu Gly Glu Met Ser Gln Pro Thr Pro Ser ProAla Pro Ala Ala Gly 370 375 380 Gly Arg Val Tyr Pro Val Ser Val Pro TyrSer Ser Ala Gln Ser Thr 385 390 395 400 Ser Lys Thr Ser Val Thr Leu SerLeu Val Met Pro Ser Gln Gly Gln 405 410 415 Met Val Asn Gly Ala His SerAla Ser Thr Leu Asp Glu Ala Thr Pro 420 425 430 Thr Leu Thr Asn Gln SerPro Thr Leu Thr Leu Gln Ser Thr Asn Thr 435 440 445 His Thr Gln Ser SerSer Ser Ser Ser Asp Gly Gly Leu Phe Arg Ser 450 455 460 Arg Pro Ala HisSer Leu Pro Pro Gly Glu Asp Gly Arg Val Glu Pro 465 470 475 480 Tyr ValAsp Phe Ala Glu Phe Tyr Arg Leu Trp Ser Val Asp His Gly 485 490 495 GluGln Ser Val Val Thr Ala Pro 500 3 2656 DNA Homo sapiens CDS(183)..(1919) 3 gtcgagatcc attgtgctct aaagacggct gtggccgctg cctctacccccgccacggat 60 cgccgggtag taggactgcg cggctccagg ctgagggtcg gtccggaggcgggtgggcgc 120 gggtctcacc cggattgtcc gggtggcacc gttcccggcc ccaccgggcgccgcgaggga 180 tc atg tct aca gcc tct gcc gcc tcc tcc tcc tcc tcg tcttcg gcc 227 Met Ser Thr Ala Ser Ala Ala Ser Ser Ser Ser Ser Ser Ser Ala1 5 10 15 ggt gag atg atc gaa gcc cct tcc cag gtc ctc aac ttt gaa gagatc 275 Gly Glu Met Ile Glu Ala Pro Ser Gln Val Leu Asn Phe Glu Glu Ile20 25 30 gac tac aag gag atc gag gtg gaa gag gtt gtt gga aga gga gcc ttt323 Asp Tyr Lys Glu Ile Glu Val Glu Glu Val Val Gly Arg Gly Ala Phe 3540 45 gga gtt gtt tgc aaa gct aag tgg aga gca aaa gat gtt gct att aaa371 Gly Val Val Cys Lys Ala Lys Trp Arg Ala Lys Asp Val Ala Ile Lys 5055 60 caa ata gaa agt gaa tct gag agg aaa gcg ttt att gta gag ctt cgg419 Gln Ile Glu Ser Glu Ser Glu Arg Lys Ala Phe Ile Val Glu Leu Arg 6570 75 cag tta tcc cgt gtg aac cat cct aat att gta aag ctt tat gga gcc467 Gln Leu Ser Arg Val Asn His Pro Asn Ile Val Lys Leu Tyr Gly Ala 8085 90 95 tgc ttg aat cca gtg tgt ctt gtg atg gaa tat gct gaa ggg ggc tct515 Cys Leu Asn Pro Val Cys Leu Val Met Glu Tyr Ala Glu Gly Gly Ser 100105 110 tta tat aat gtg ctg cat ggt gct gaa cca ttg cca tat tat act gct563 Leu Tyr Asn Val Leu His Gly Ala Glu Pro Leu Pro Tyr Tyr Thr Ala 115120 125 gcc cac gca atg agt tgg tgt tta cag tgt tcc caa gga gtg gct tat611 Ala His Ala Met Ser Trp Cys Leu Gln Cys Ser Gln Gly Val Ala Tyr 130135 140 ctt cac agc atg caa ccc aaa gcg cta att cac agg gac ctg aaa cca659 Leu His Ser Met Gln Pro Lys Ala Leu Ile His Arg Asp Leu Lys Pro 145150 155 cca aac tta ctg ctg gtt gca ggg ggg aca gtt cta aaa att tgt gat707 Pro Asn Leu Leu Leu Val Ala Gly Gly Thr Val Leu Lys Ile Cys Asp 160165 170 175 ttt ggt aca gcc tgt gac att cag aca cac atg acc aat aac aagggg 755 Phe Gly Thr Ala Cys Asp Ile Gln Thr His Met Thr Asn Asn Lys Gly180 185 190 agt gct gct tgg atg gca cct gaa gtt ttt gaa ggt agt aat tacagt 803 Ser Ala Ala Trp Met Ala Pro Glu Val Phe Glu Gly Ser Asn Tyr Ser195 200 205 gaa aaa tgt gac gtc ttc agc tgg ggt att att ctt tgg gaa gtgata 851 Glu Lys Cys Asp Val Phe Ser Trp Gly Ile Ile Leu Trp Glu Val Ile210 215 220 acg cgt cgg aaa ccc ttt gat gag att ggt ggc cca gct ttc cgaatc 899 Thr Arg Arg Lys Pro Phe Asp Glu Ile Gly Gly Pro Ala Phe Arg Ile225 230 235 atg tgg gct gtt cat aat ggt act cga cca cca ctg ata aaa aattta 947 Met Trp Ala Val His Asn Gly Thr Arg Pro Pro Leu Ile Lys Asn Leu240 245 250 255 cct aag ccc att gag agc ctg atg act cgt tgt tgg tct aaagat cct 995 Pro Lys Pro Ile Glu Ser Leu Met Thr Arg Cys Trp Ser Lys AspPro 260 265 270 tcc cag cgc cct tca atg gag gaa att gtg aaa ata atg actcac ttg 1043 Ser Gln Arg Pro Ser Met Glu Glu Ile Val Lys Ile Met Thr HisLeu 275 280 285 atg cgg tac ttt cca gga gca gat gag cca tta cag tat ccttgt cag 1091 Met Arg Tyr Phe Pro Gly Ala Asp Glu Pro Leu Gln Tyr Pro CysGln 290 295 300 tat tca gat gaa gga cag agc aac tct gcc acc agt aca ggctca ttc 1139 Tyr Ser Asp Glu Gly Gln Ser Asn Ser Ala Thr Ser Thr Gly SerPhe 305 310 315 atg gac att gct tct aca aat acg agt aac aaa agt gac actaat atg 1187 Met Asp Ile Ala Ser Thr Asn Thr Ser Asn Lys Ser Asp Thr AsnMet 320 325 330 335 gag caa gtt cct gcc aca aat gat act att aag cgc ttagaa tca aaa 1235 Glu Gln Val Pro Ala Thr Asn Asp Thr Ile Lys Arg Leu GluSer Lys 340 345 350 ttg ttg aaa aat cag gca aag caa cag agt gaa tct ggacgt tta agc 1283 Leu Leu Lys Asn Gln Ala Lys Gln Gln Ser Glu Ser Gly ArgLeu Ser 355 360 365 ttg gga gcc tcc cat ggg agc agt gtg gag agc ttg ccccca acc tct 1331 Leu Gly Ala Ser His Gly Ser Ser Val Glu Ser Leu Pro ProThr Ser 370 375 380 gag ggc aag agg atg agt gct gac atg tct gaa ata gaagct agg atc 1379 Glu Gly Lys Arg Met Ser Ala Asp Met Ser Glu Ile Glu AlaArg Ile 385 390 395 gcc gca acc aca ggc aac gga cag cca aga cgt aga tccatc caa gac 1427 Ala Ala Thr Thr Gly Asn Gly Gln Pro Arg Arg Arg Ser IleGln Asp 400 405 410 415 ttg act gta act gga aca gaa cct ggt cag gtg agcagt agg tca tcc 1475 Leu Thr Val Thr Gly Thr Glu Pro Gly Gln Val Ser SerArg Ser Ser 420 425 430 agt ccc agt gtc aga atg att act acc tca gga ccaacc tca gaa aag 1523 Ser Pro Ser Val Arg Met Ile Thr Thr Ser Gly Pro ThrSer Glu Lys 435 440 445 cca act cga agt cat cca tgg acc cct gat gat tccaca gat acc aat 1571 Pro Thr Arg Ser His Pro Trp Thr Pro Asp Asp Ser ThrAsp Thr Asn 450 455 460 gga tca gat aac tcc atc cca atg gct tat ctt acactg gat cac caa 1619 Gly Ser Asp Asn Ser Ile Pro Met Ala Tyr Leu Thr LeuAsp His Gln 465 470 475 cta cag cct cta gca ccg tgc cca aac tcc aaa gaatct atg gca gtg 1667 Leu Gln Pro Leu Ala Pro Cys Pro Asn Ser Lys Glu SerMet Ala Val 480 485 490 495 ttt gaa cag cat tgt aaa atg gca caa gaa tatatg aaa gtt caa aca 1715 Phe Glu Gln His Cys Lys Met Ala Gln Glu Tyr MetLys Val Gln Thr 500 505 510 gaa att gca ttg tta tta cag aga aag caa gaacta gtt gca gaa ctg 1763 Glu Ile Ala Leu Leu Leu Gln Arg Lys Gln Glu LeuVal Ala Glu Leu 515 520 525 gac cag gat gaa aag gac cag caa aat aca tctcgc ctg gta cag gaa 1811 Asp Gln Asp Glu Lys Asp Gln Gln Asn Thr Ser ArgLeu Val Gln Glu 530 535 540 cat aaa aag ctt tta gat gaa aac aaa agc ctttct act tac tac cag 1859 His Lys Lys Leu Leu Asp Glu Asn Lys Ser Leu SerThr Tyr Tyr Gln 545 550 555 caa tgc aaa aaa caa cta gag gtc atc aga agtcag cag cag aaa cga 1907 Gln Cys Lys Lys Gln Leu Glu Val Ile Arg Ser GlnGln Gln Lys Arg 560 565 570 575 caa ggc act tca tgattctctg ggaccgttacattttgaaat atgcaaagaa 1959 Gln Gly Thr Ser agactttttt tttaaggaaaggaaaacctt ataatgacga ttcatgagtg ttagcttttt 2019 ggcgtgttct gaatgccaactgcctatatt tgctgcattt ttttcattgt ttattttcct 2079 tttctcatgg tggacatacaattttactgt ttcattgcat aacatggtag catctgtgac 2139 ttgaatgagc agcactttgcaacttcaaaa cagatgcagt gaactgtggc tgtatatgca 2199 tgctcattgt gtgaaggctagcctaacaga acaggaggta tcaaactagc tgctatgtgc 2259 aaacagcgtc cattttttcatattagaggt ggaacctcaa gaatgacttt attcttgtat 2319 ctcatctcaa aatattaataatttttttcc caaaagatgg tatataccaa gttaaagaca 2379 gggtattata aatttagagtgattggtggt atattacgga aatacggaac ctttagggat 2439 agttccgtgt aagggctttgatgccagcat ccttggatca gtactgaact cagttccatc 2499 cgtaaaatat gtaaaggtaagtggcagctg ctctatttaa tgaaagcagt tttaccggat 2559 tttgttagac taaaatttgattgtgataca ttgaacaaaa tggaactcat tttttttaag 2619 gagtaaagat tttctttagagcacaatgga tctcgac 2656 4 579 PRT Homo sapiens 4 Met Ser Thr Ala Ser AlaAla Ser Ser Ser Ser Ser Ser Ser Ala Gly 1 5 10 15 Glu Met Ile Glu AlaPro Ser Gln Val Leu Asn Phe Glu Glu Ile Asp 20 25 30 Tyr Lys Glu Ile GluVal Glu Glu Val Val Gly Arg Gly Ala Phe Gly 35 40 45 Val Val Cys Lys AlaLys Trp Arg Ala Lys Asp Val Ala Ile Lys Gln 50 55 60 Ile Glu Ser Glu SerGlu Arg Lys Ala Phe Ile Val Glu Leu Arg Gln 65 70 75 80 Leu Ser Arg ValAsn His Pro Asn Ile Val Lys Leu Tyr Gly Ala Cys 85 90 95 Leu Asn Pro ValCys Leu Val Met Glu Tyr Ala Glu Gly Gly Ser Leu 100 105 110 Tyr Asn ValLeu His Gly Ala Glu Pro Leu Pro Tyr Tyr Thr Ala Ala 115 120 125 His AlaMet Ser Trp Cys Leu Gln Cys Ser Gln Gly Val Ala Tyr Leu 130 135 140 HisSer Met Gln Pro Lys Ala Leu Ile His Arg Asp Leu Lys Pro Pro 145 150 155160 Asn Leu Leu Leu Val Ala Gly Gly Thr Val Leu Lys Ile Cys Asp Phe 165170 175 Gly Thr Ala Cys Asp Ile Gln Thr His Met Thr Asn Asn Lys Gly Ser180 185 190 Ala Ala Trp Met Ala Pro Glu Val Phe Glu Gly Ser Asn Tyr SerGlu 195 200 205 Lys Cys Asp Val Phe Ser Trp Gly Ile Ile Leu Trp Glu ValIle Thr 210 215 220 Arg Arg Lys Pro Phe Asp Glu Ile Gly Gly Pro Ala PheArg Ile Met 225 230 235 240 Trp Ala Val His Asn Gly Thr Arg Pro Pro LeuIle Lys Asn Leu Pro 245 250 255 Lys Pro Ile Glu Ser Leu Met Thr Arg CysTrp Ser Lys Asp Pro Ser 260 265 270 Gln Arg Pro Ser Met Glu Glu Ile ValLys Ile Met Thr His Leu Met 275 280 285 Arg Tyr Phe Pro Gly Ala Asp GluPro Leu Gln Tyr Pro Cys Gln Tyr 290 295 300 Ser Asp Glu Gly Gln Ser AsnSer Ala Thr Ser Thr Gly Ser Phe Met 305 310 315 320 Asp Ile Ala Ser ThrAsn Thr Ser Asn Lys Ser Asp Thr Asn Met Glu 325 330 335 Gln Val Pro AlaThr Asn Asp Thr Ile Lys Arg Leu Glu Ser Lys Leu 340 345 350 Leu Lys AsnGln Ala Lys Gln Gln Ser Glu Ser Gly Arg Leu Ser Leu 355 360 365 Gly AlaSer His Gly Ser Ser Val Glu Ser Leu Pro Pro Thr Ser Glu 370 375 380 GlyLys Arg Met Ser Ala Asp Met Ser Glu Ile Glu Ala Arg Ile Ala 385 390 395400 Ala Thr Thr Gly Asn Gly Gln Pro Arg Arg Arg Ser Ile Gln Asp Leu 405410 415 Thr Val Thr Gly Thr Glu Pro Gly Gln Val Ser Ser Arg Ser Ser Ser420 425 430 Pro Ser Val Arg Met Ile Thr Thr Ser Gly Pro Thr Ser Glu LysPro 435 440 445 Thr Arg Ser His Pro Trp Thr Pro Asp Asp Ser Thr Asp ThrAsn Gly 450 455 460 Ser Asp Asn Ser Ile Pro Met Ala Tyr Leu Thr Leu AspHis Gln Leu 465 470 475 480 Gln Pro Leu Ala Pro Cys Pro Asn Ser Lys GluSer Met Ala Val Phe 485 490 495 Glu Gln His Cys Lys Met Ala Gln Glu TyrMet Lys Val Gln Thr Glu 500 505 510 Ile Ala Leu Leu Leu Gln Arg Lys GlnGlu Leu Val Ala Glu Leu Asp 515 520 525 Gln Asp Glu Lys Asp Gln Gln AsnThr Ser Arg Leu Val Gln Glu His 530 535 540 Lys Lys Leu Leu Asp Glu AsnLys Ser Leu Ser Thr Tyr Tyr Gln Gln 545 550 555 560 Cys Lys Lys Gln LeuGlu Val Ile Arg Ser Gln Gln Gln Lys Arg Gln 565 570 575 Gly Thr Ser 5 8PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 5 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 6 6 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 6 His HisHis His His His 1 5 7 27 DNA Artificial Sequence Description ofArtificial Sequence Synthetic DNA 7 ccggaattca tggcggcgca gaggagg 27 872 DNA Artificial Sequence Description of Artificial Sequence SyntheticDNA 8 agctctagat cattatttat cgtcatcgtc tttgtagtca gaacctccgg tacccggtgc60 tgtcaccacg ct 72 9 5 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 9 Gly Thr Gly Gly Ser 1 5 10 1569DNA Homo sapiens CDS (7)..(1557) 10 gaattc atg gcg gcg cag agg agg agcttg ctg cag agt gag cag cag 48 Met Ala Ala Gln Arg Arg Ser Leu Leu GlnSer Glu Gln Gln 1 5 10 cca agc tgg aca gat gac ctg cct ctc tgc cac ctctct ggg gtt ggc 96 Pro Ser Trp Thr Asp Asp Leu Pro Leu Cys His Leu SerGly Val Gly 15 20 25 30 tca gcc tcc aac cgc agc tac tct gct gat ggc aagggc act gag agc 144 Ser Ala Ser Asn Arg Ser Tyr Ser Ala Asp Gly Lys GlyThr Glu Ser 35 40 45 cac ccg cca gag gac agc tgg ctc aag ttc agg agt gagaac aac tgc 192 His Pro Pro Glu Asp Ser Trp Leu Lys Phe Arg Ser Glu AsnAsn Cys 50 55 60 ttc ctg tat ggg gtc ttc aac ggc tat gat ggc aac cga gtgacc aac 240 Phe Leu Tyr Gly Val Phe Asn Gly Tyr Asp Gly Asn Arg Val ThrAsn 65 70 75 ttc gtg gcc cag cgg ctg tcc gca gag ctc ctg ctg ggc cag ctgaat 288 Phe Val Ala Gln Arg Leu Ser Ala Glu Leu Leu Leu Gly Gln Leu Asn80 85 90 gcc gag cac gcc gag gcc gat gtg cgg cgt gtg ctg ctg cag gcc ttc336 Ala Glu His Ala Glu Ala Asp Val Arg Arg Val Leu Leu Gln Ala Phe 95100 105 110 gat gtg gtg gag agg agc ttc ctg gag tcc att gac gac gcc ttggct 384 Asp Val Val Glu Arg Ser Phe Leu Glu Ser Ile Asp Asp Ala Leu Ala115 120 125 gag aag gca agc ctc cag tcg caa ttg cca gag gga gtc cct cagcac 432 Glu Lys Ala Ser Leu Gln Ser Gln Leu Pro Glu Gly Val Pro Gln His130 135 140 cag ctg cct cct cag tat cag aag atc ctt gag aga ctc aag acgtta 480 Gln Leu Pro Pro Gln Tyr Gln Lys Ile Leu Glu Arg Leu Lys Thr Leu145 150 155 gag agg gaa att tcg gga ggg gcc atg gcc gtt gtg gcg gtc cttctc 528 Glu Arg Glu Ile Ser Gly Gly Ala Met Ala Val Val Ala Val Leu Leu160 165 170 aac aac aag ctc tac gtc gcc aat gtc ggt aca aac cgt gca ctttta 576 Asn Asn Lys Leu Tyr Val Ala Asn Val Gly Thr Asn Arg Ala Leu Leu175 180 185 190 tgc aaa tcg aca gtg gat ggg ttg cag gtg aca cag ctg aacgtg gac 624 Cys Lys Ser Thr Val Asp Gly Leu Gln Val Thr Gln Leu Asn ValAsp 195 200 205 cac acc aca gag aac gag gat gag ctc ttc cgt ctt tcg cagctg ggc 672 His Thr Thr Glu Asn Glu Asp Glu Leu Phe Arg Leu Ser Gln LeuGly 210 215 220 ttg gat gct gga aag atc aag cag gtg ggg atc atc tgt gggcag gag 720 Leu Asp Ala Gly Lys Ile Lys Gln Val Gly Ile Ile Cys Gly GlnGlu 225 230 235 agc acc cgg cgg atc ggg gat tac aag gtt aaa tat ggc tacacg gac 768 Ser Thr Arg Arg Ile Gly Asp Tyr Lys Val Lys Tyr Gly Tyr ThrAsp 240 245 250 att gac ctt ctc agc gct gcc aag tcc aaa cca atc atc gcagag cca 816 Ile Asp Leu Leu Ser Ala Ala Lys Ser Lys Pro Ile Ile Ala GluPro 255 260 265 270 gaa atc cat ggg gca cag ccg ctg gat ggg gtg acg ggcttc ttg gtg 864 Glu Ile His Gly Ala Gln Pro Leu Asp Gly Val Thr Gly PheLeu Val 275 280 285 ctg atg tcg gag ggg ttg tac aag gcc cta gag gca gcccat ggg cct 912 Leu Met Ser Glu Gly Leu Tyr Lys Ala Leu Glu Ala Ala HisGly Pro 290 295 300 ggg cag gcc aac cag gag att gct gcg atg att gac actgag ttt gcc 960 Gly Gln Ala Asn Gln Glu Ile Ala Ala Met Ile Asp Thr GluPhe Ala 305 310 315 aag cag acc tcc ctg gac gca gtg gcc cag gcc gtc gtggac cgg gtg 1008 Lys Gln Thr Ser Leu Asp Ala Val Ala Gln Ala Val Val AspArg Val 320 325 330 aag cgc atc cac agc gac acc ttc gcc agt ggt ggg gagcgt gcc agg 1056 Lys Arg Ile His Ser Asp Thr Phe Ala Ser Gly Gly Glu ArgAla Arg 335 340 345 350 ttc tgc ccc cgg cac gag gac atg acc ctg cta gtgagg aac ttt ggc 1104 Phe Cys Pro Arg His Glu Asp Met Thr Leu Leu Val ArgAsn Phe Gly 355 360 365 tac ccg ctg ggc gaa atg agc cag ccc aca ccg agccca gcc cca gct 1152 Tyr Pro Leu Gly Glu Met Ser Gln Pro Thr Pro Ser ProAla Pro Ala 370 375 380 gca gga gga cga gtg tac cct gtg tct gtg cca tactcc agc gcc cag 1200 Ala Gly Gly Arg Val Tyr Pro Val Ser Val Pro Tyr SerSer Ala Gln 385 390 395 agc acc agc aag acc agc gtg acc ctc tcc ctt gtcatg ccc tcc cag 1248 Ser Thr Ser Lys Thr Ser Val Thr Leu Ser Leu Val MetPro Ser Gln 400 405 410 ggc cag atg gtc aac ggg gct cac agt gct tcc accctg gac gaa gcc 1296 Gly Gln Met Val Asn Gly Ala His Ser Ala Ser Thr LeuAsp Glu Ala 415 420 425 430 acc ccc acc ctc acc aac caa agc ccg acc ttaacc ctg cag tcc acc 1344 Thr Pro Thr Leu Thr Asn Gln Ser Pro Thr Leu ThrLeu Gln Ser Thr 435 440 445 aac acg cac acg cag agc agc agc tcc agc tctgac gga ggc ctc ttc 1392 Asn Thr His Thr Gln Ser Ser Ser Ser Ser Ser AspGly Gly Leu Phe 450 455 460 cgc tcc cgg ccc gcc cac tcg ctc ccg cct ggcgag gac ggt cgt gtt 1440 Arg Ser Arg Pro Ala His Ser Leu Pro Pro Gly GluAsp Gly Arg Val 465 470 475 gag ccc tat gtg gac ttt gct gag ttt tac cgcctc tgg agc gtg gac 1488 Glu Pro Tyr Val Asp Phe Ala Glu Phe Tyr Arg LeuTrp Ser Val Asp 480 485 490 cat ggc gag cag agc gtg gtg aca gca ccg ggtacc gga ggt tct gac 1536 His Gly Glu Gln Ser Val Val Thr Ala Pro Gly ThrGly Gly Ser Asp 495 500 505 510 tac aaa gac gat gac gat aaa taatgatctaga 1569 Tyr Lys Asp Asp Asp Asp Lys 515 11 517 PRT Homo sapiens 11 MetAla Ala Gln Arg Arg Ser Leu Leu Gln Ser Glu Gln Gln Pro Ser 1 5 10 15Trp Thr Asp Asp Leu Pro Leu Cys His Leu Ser Gly Val Gly Ser Ala 20 25 30Ser Asn Arg Ser Tyr Ser Ala Asp Gly Lys Gly Thr Glu Ser His Pro 35 40 45Pro Glu Asp Ser Trp Leu Lys Phe Arg Ser Glu Asn Asn Cys Phe Leu 50 55 60Tyr Gly Val Phe Asn Gly Tyr Asp Gly Asn Arg Val Thr Asn Phe Val 65 70 7580 Ala Gln Arg Leu Ser Ala Glu Leu Leu Leu Gly Gln Leu Asn Ala Glu 85 9095 His Ala Glu Ala Asp Val Arg Arg Val Leu Leu Gln Ala Phe Asp Val 100105 110 Val Glu Arg Ser Phe Leu Glu Ser Ile Asp Asp Ala Leu Ala Glu Lys115 120 125 Ala Ser Leu Gln Ser Gln Leu Pro Glu Gly Val Pro Gln His GlnLeu 130 135 140 Pro Pro Gln Tyr Gln Lys Ile Leu Glu Arg Leu Lys Thr LeuGlu Arg 145 150 155 160 Glu Ile Ser Gly Gly Ala Met Ala Val Val Ala ValLeu Leu Asn Asn 165 170 175 Lys Leu Tyr Val Ala Asn Val Gly Thr Asn ArgAla Leu Leu Cys Lys 180 185 190 Ser Thr Val Asp Gly Leu Gln Val Thr GlnLeu Asn Val Asp His Thr 195 200 205 Thr Glu Asn Glu Asp Glu Leu Phe ArgLeu Ser Gln Leu Gly Leu Asp 210 215 220 Ala Gly Lys Ile Lys Gln Val GlyIle Ile Cys Gly Gln Glu Ser Thr 225 230 235 240 Arg Arg Ile Gly Asp TyrLys Val Lys Tyr Gly Tyr Thr Asp Ile Asp 245 250 255 Leu Leu Ser Ala AlaLys Ser Lys Pro Ile Ile Ala Glu Pro Glu Ile 260 265 270 His Gly Ala GlnPro Leu Asp Gly Val Thr Gly Phe Leu Val Leu Met 275 280 285 Ser Glu GlyLeu Tyr Lys Ala Leu Glu Ala Ala His Gly Pro Gly Gln 290 295 300 Ala AsnGln Glu Ile Ala Ala Met Ile Asp Thr Glu Phe Ala Lys Gln 305 310 315 320Thr Ser Leu Asp Ala Val Ala Gln Ala Val Val Asp Arg Val Lys Arg 325 330335 Ile His Ser Asp Thr Phe Ala Ser Gly Gly Glu Arg Ala Arg Phe Cys 340345 350 Pro Arg His Glu Asp Met Thr Leu Leu Val Arg Asn Phe Gly Tyr Pro355 360 365 Leu Gly Glu Met Ser Gln Pro Thr Pro Ser Pro Ala Pro Ala AlaGly 370 375 380 Gly Arg Val Tyr Pro Val Ser Val Pro Tyr Ser Ser Ala GlnSer Thr 385 390 395 400 Ser Lys Thr Ser Val Thr Leu Ser Leu Val Met ProSer Gln Gly Gln 405 410 415 Met Val Asn Gly Ala His Ser Ala Ser Thr LeuAsp Glu Ala Thr Pro 420 425 430 Thr Leu Thr Asn Gln Ser Pro Thr Leu ThrLeu Gln Ser Thr Asn Thr 435 440 445 His Thr Gln Ser Ser Ser Ser Ser SerAsp Gly Gly Leu Phe Arg Ser 450 455 460 Arg Pro Ala His Ser Leu Pro ProGly Glu Asp Gly Arg Val Glu Pro 465 470 475 480 Tyr Val Asp Phe Ala GluPhe Tyr Arg Leu Trp Ser Val Asp His Gly 485 490 495 Glu Gln Ser Val ValThr Ala Pro Gly Thr Gly Gly Ser Asp Tyr Lys 500 505 510 Asp Asp Asp AspLys 515 12 27 DNA Artificial Sequence Description of Artificial SequenceSynthetic DNA 12 ccggaattca tgtctacagc ctctgcc 27 13 66 DNA ArtificialSequence Description of Artificial Sequence Synthetic DNA 13 agctctagatcattagtgat ggtgatggtg atgagatcca ccggtacctg aagtgccttg 60 tcgttt 66 141788 DNA Homo sapiens CDS (7)..(1776) 14 gaattc atg tct aca gcc tct gccgcc tcc tcc tcc tcc tcg tct tcg 48 Met Ser Thr Ala Ser Ala Ala Ser SerSer Ser Ser Ser Ser 1 5 10 gcc ggt gag atg atc gaa gcc cct tcc cag gtcctc aac ttt gaa gag 96 Ala Gly Glu Met Ile Glu Ala Pro Ser Gln Val LeuAsn Phe Glu Glu 15 20 25 30 atc gac tac aag gag atc gag gtg gaa gag gttgtt gga aga gga gcc 144 Ile Asp Tyr Lys Glu Ile Glu Val Glu Glu Val ValGly Arg Gly Ala 35 40 45 ttt gga gtt gtt tgc aaa gct aag tgg aga gca aaagat gtt gct att 192 Phe Gly Val Val Cys Lys Ala Lys Trp Arg Ala Lys AspVal Ala Ile 50 55 60 aaa caa ata gaa agt gaa tct gag agg aaa gcg ttt attgta gag ctt 240 Lys Gln Ile Glu Ser Glu Ser Glu Arg Lys Ala Phe Ile ValGlu Leu 65 70 75 cgg cag tta tcc cgt gtg aac cat cct aat att gta aag ctttat gga 288 Arg Gln Leu Ser Arg Val Asn His Pro Asn Ile Val Lys Leu TyrGly 80 85 90 gcc tgc ttg aat cca gtg tgt ctt gtg atg gaa tat gct gaa gggggc 336 Ala Cys Leu Asn Pro Val Cys Leu Val Met Glu Tyr Ala Glu Gly Gly95 100 105 110 tct tta tat aat gtg ctg cat ggt gct gaa cca ttg cca tattat act 384 Ser Leu Tyr Asn Val Leu His Gly Ala Glu Pro Leu Pro Tyr TyrThr 115 120 125 gct gcc cac gca atg agt tgg tgt tta cag tgt tcc caa ggagtg gct 432 Ala Ala His Ala Met Ser Trp Cys Leu Gln Cys Ser Gln Gly ValAla 130 135 140 tat ctt cac agc atg caa ccc aaa gcg cta att cac agg gacctg aaa 480 Tyr Leu His Ser Met Gln Pro Lys Ala Leu Ile His Arg Asp LeuLys 145 150 155 cca cca aac tta ctg ctg gtt gca ggg ggg aca gtt cta aaaatt tgt 528 Pro Pro Asn Leu Leu Leu Val Ala Gly Gly Thr Val Leu Lys IleCys 160 165 170 gat ttt ggt aca gcc tgt gac att cag aca cac atg acc aataac aag 576 Asp Phe Gly Thr Ala Cys Asp Ile Gln Thr His Met Thr Asn AsnLys 175 180 185 190 ggg agt gct gct tgg atg gca cct gaa gtt ttt gaa ggtagt aat tac 624 Gly Ser Ala Ala Trp Met Ala Pro Glu Val Phe Glu Gly SerAsn Tyr 195 200 205 agt gaa aaa tgt gac gtc ttc agc tgg ggt att att ctttgg gaa gtg 672 Ser Glu Lys Cys Asp Val Phe Ser Trp Gly Ile Ile Leu TrpGlu Val 210 215 220 ata acg cgt cgg aaa ccc ttt gat gag att ggt ggc ccagct ttc cga 720 Ile Thr Arg Arg Lys Pro Phe Asp Glu Ile Gly Gly Pro AlaPhe Arg 225 230 235 atc atg tgg gct gtt cat aat ggt act cga cca cca ctgata aaa aat 768 Ile Met Trp Ala Val His Asn Gly Thr Arg Pro Pro Leu IleLys Asn 240 245 250 tta cct aag ccc att gag agc ctg atg act cgt tgt tggtct aaa gat 816 Leu Pro Lys Pro Ile Glu Ser Leu Met Thr Arg Cys Trp SerLys Asp 255 260 265 270 cct tcc cag cgc cct tca atg gag gaa att gtg aaaata atg act cac 864 Pro Ser Gln Arg Pro Ser Met Glu Glu Ile Val Lys IleMet Thr His 275 280 285 ttg atg cgg tac ttt cca gga gca gat gag cca ttacag tat cct tgt 912 Leu Met Arg Tyr Phe Pro Gly Ala Asp Glu Pro Leu GlnTyr Pro Cys 290 295 300 cag tat tca gat gaa gga cag agc aac tct gcc accagt aca ggc tca 960 Gln Tyr Ser Asp Glu Gly Gln Ser Asn Ser Ala Thr SerThr Gly Ser 305 310 315 ttc atg gac att gct tct aca aat acg agt aac aaaagt gac act aat 1008 Phe Met Asp Ile Ala Ser Thr Asn Thr Ser Asn Lys SerAsp Thr Asn 320 325 330 atg gag caa gtt cct gcc aca aat gat act att aagcgc tta gaa tca 1056 Met Glu Gln Val Pro Ala Thr Asn Asp Thr Ile Lys ArgLeu Glu Ser 335 340 345 350 aaa ttg ttg aaa aat cag gca aag caa cag agtgaa tct gga cgt tta 1104 Lys Leu Leu Lys Asn Gln Ala Lys Gln Gln Ser GluSer Gly Arg Leu 355 360 365 agc ttg gga gcc tcc cat ggg agc agt gtg gagagc ttg ccc cca acc 1152 Ser Leu Gly Ala Ser His Gly Ser Ser Val Glu SerLeu Pro Pro Thr 370 375 380 tct gag ggc aag agg atg agt gct gac atg tctgaa ata gaa gct agg 1200 Ser Glu Gly Lys Arg Met Ser Ala Asp Met Ser GluIle Glu Ala Arg 385 390 395 atc gcc gca acc aca ggc aac gga cag cca agacgt aga tcc atc caa 1248 Ile Ala Ala Thr Thr Gly Asn Gly Gln Pro Arg ArgArg Ser Ile Gln 400 405 410 gac ttg act gta act gga aca gaa cct ggt caggtg agc agt agg tca 1296 Asp Leu Thr Val Thr Gly Thr Glu Pro Gly Gln ValSer Ser Arg Ser 415 420 425 430 tcc agt ccc agt gtc aga atg att act acctca gga cca acc tca gaa 1344 Ser Ser Pro Ser Val Arg Met Ile Thr Thr SerGly Pro Thr Ser Glu 435 440 445 aag cca act cga agt cat cca tgg acc cctgat gat tcc aca gat acc 1392 Lys Pro Thr Arg Ser His Pro Trp Thr Pro AspAsp Ser Thr Asp Thr 450 455 460 aat gga tca gat aac tcc atc cca atg gcttat ctt aca ctg gat cac 1440 Asn Gly Ser Asp Asn Ser Ile Pro Met Ala TyrLeu Thr Leu Asp His 465 470 475 caa cta cag cct cta gca ccg tgc cca aactcc aaa gaa tct atg gca 1488 Gln Leu Gln Pro Leu Ala Pro Cys Pro Asn SerLys Glu Ser Met Ala 480 485 490 gtg ttt gaa cag cat tgt aaa atg gca caagaa tat atg aaa gtt caa 1536 Val Phe Glu Gln His Cys Lys Met Ala Gln GluTyr Met Lys Val Gln 495 500 505 510 aca gaa att gca ttg tta tta cag agaaag caa gaa cta gtt gca gaa 1584 Thr Glu Ile Ala Leu Leu Leu Gln Arg LysGln Glu Leu Val Ala Glu 515 520 525 ctg gac cag gat gaa aag gac cag caaaat aca tct cgc ctg gta cag 1632 Leu Asp Gln Asp Glu Lys Asp Gln Gln AsnThr Ser Arg Leu Val Gln 530 535 540 gaa cat aaa aag ctt tta gat gaa aacaaa agc ctt tct act tac tac 1680 Glu His Lys Lys Leu Leu Asp Glu Asn LysSer Leu Ser Thr Tyr Tyr 545 550 555 cag caa tgc aaa aaa caa cta gag gtcatc aga agt cag cag cag aaa 1728 Gln Gln Cys Lys Lys Gln Leu Glu Val IleArg Ser Gln Gln Gln Lys 560 565 570 cga caa ggc act tca ggt acc ggt ggatct cat cac cat cac cat cac 1776 Arg Gln Gly Thr Ser Gly Thr Gly Gly SerHis His His His His His 575 580 585 590 taatgatcta ga 1788 15 590 PRTHomo sapiens 15 Met Ser Thr Ala Ser Ala Ala Ser Ser Ser Ser Ser Ser SerAla Gly 1 5 10 15 Glu Met Ile Glu Ala Pro Ser Gln Val Leu Asn Phe GluGlu Ile Asp 20 25 30 Tyr Lys Glu Ile Glu Val Glu Glu Val Val Gly Arg GlyAla Phe Gly 35 40 45 Val Val Cys Lys Ala Lys Trp Arg Ala Lys Asp Val AlaIle Lys Gln 50 55 60 Ile Glu Ser Glu Ser Glu Arg Lys Ala Phe Ile Val GluLeu Arg Gln 65 70 75 80 Leu Ser Arg Val Asn His Pro Asn Ile Val Lys LeuTyr Gly Ala Cys 85 90 95 Leu Asn Pro Val Cys Leu Val Met Glu Tyr Ala GluGly Gly Ser Leu 100 105 110 Tyr Asn Val Leu His Gly Ala Glu Pro Leu ProTyr Tyr Thr Ala Ala 115 120 125 His Ala Met Ser Trp Cys Leu Gln Cys SerGln Gly Val Ala Tyr Leu 130 135 140 His Ser Met Gln Pro Lys Ala Leu IleHis Arg Asp Leu Lys Pro Pro 145 150 155 160 Asn Leu Leu Leu Val Ala GlyGly Thr Val Leu Lys Ile Cys Asp Phe 165 170 175 Gly Thr Ala Cys Asp IleGln Thr His Met Thr Asn Asn Lys Gly Ser 180 185 190 Ala Ala Trp Met AlaPro Glu Val Phe Glu Gly Ser Asn Tyr Ser Glu 195 200 205 Lys Cys Asp ValPhe Ser Trp Gly Ile Ile Leu Trp Glu Val Ile Thr 210 215 220 Arg Arg LysPro Phe Asp Glu Ile Gly Gly Pro Ala Phe Arg Ile Met 225 230 235 240 TrpAla Val His Asn Gly Thr Arg Pro Pro Leu Ile Lys Asn Leu Pro 245 250 255Lys Pro Ile Glu Ser Leu Met Thr Arg Cys Trp Ser Lys Asp Pro Ser 260 265270 Gln Arg Pro Ser Met Glu Glu Ile Val Lys Ile Met Thr His Leu Met 275280 285 Arg Tyr Phe Pro Gly Ala Asp Glu Pro Leu Gln Tyr Pro Cys Gln Tyr290 295 300 Ser Asp Glu Gly Gln Ser Asn Ser Ala Thr Ser Thr Gly Ser PheMet 305 310 315 320 Asp Ile Ala Ser Thr Asn Thr Ser Asn Lys Ser Asp ThrAsn Met Glu 325 330 335 Gln Val Pro Ala Thr Asn Asp Thr Ile Lys Arg LeuGlu Ser Lys Leu 340 345 350 Leu Lys Asn Gln Ala Lys Gln Gln Ser Glu SerGly Arg Leu Ser Leu 355 360 365 Gly Ala Ser His Gly Ser Ser Val Glu SerLeu Pro Pro Thr Ser Glu 370 375 380 Gly Lys Arg Met Ser Ala Asp Met SerGlu Ile Glu Ala Arg Ile Ala 385 390 395 400 Ala Thr Thr Gly Asn Gly GlnPro Arg Arg Arg Ser Ile Gln Asp Leu 405 410 415 Thr Val Thr Gly Thr GluPro Gly Gln Val Ser Ser Arg Ser Ser Ser 420 425 430 Pro Ser Val Arg MetIle Thr Thr Ser Gly Pro Thr Ser Glu Lys Pro 435 440 445 Thr Arg Ser HisPro Trp Thr Pro Asp Asp Ser Thr Asp Thr Asn Gly 450 455 460 Ser Asp AsnSer Ile Pro Met Ala Tyr Leu Thr Leu Asp His Gln Leu 465 470 475 480 GlnPro Leu Ala Pro Cys Pro Asn Ser Lys Glu Ser Met Ala Val Phe 485 490 495Glu Gln His Cys Lys Met Ala Gln Glu Tyr Met Lys Val Gln Thr Glu 500 505510 Ile Ala Leu Leu Leu Gln Arg Lys Gln Glu Leu Val Ala Glu Leu Asp 515520 525 Gln Asp Glu Lys Asp Gln Gln Asn Thr Ser Arg Leu Val Gln Glu His530 535 540 Lys Lys Leu Leu Asp Glu Asn Lys Ser Leu Ser Thr Tyr Tyr GlnGln 545 550 555 560 Cys Lys Lys Gln Leu Glu Val Ile Arg Ser Gln Gln GlnLys Arg Gln 565 570 575 Gly Thr Ser Gly Thr Gly Gly Ser His His His HisHis His 580 585 590 16 41 DNA Artificial Sequence Description ofArtificial Sequence Synthetic DNA 16 ttctgaaggg cttccaccct ggacgaagccacccccaccc t 41 17 69 DNA Artificial Sequence Description of ArtificialSequence Synthetic DNA 17 tataagcttt tattatttat cgtcatcgtc tttgtagtccggtgctgtca ccacgctctg 60 ctcgccatg 69 18 34 DNA Artificial SequenceDescription of Artificial Sequence Synthetic DNA 18 ccggaattccaccatggagc ttcggcagtt atcc 34 19 28 DNA Artificial Sequence Descriptionof Artificial Sequence Synthetic DNA 19 ccggaattcc tactgacaag gatactgt28 20 19 DNA Artificial Sequence Description of Artificial SequenceSynthetic DNA 20 tcttcagctg gggtattat 19 21 20 DNA Artificial SequenceDescription of Artificial Sequence Synthetic DNA 21 gctttatttccatgctgggc 20 22 27 DNA Artificial Sequence Description of ArtificialSequence Synthetic DNA 22 cggaattcga gctccggcag tgtcgcg 27 23 30 DNAArtificial Sequence Description of Artificial Sequence Synthetic DNA 23aactgcaggc tactgacaag gatactgtaa 30 24 31 DNA Artificial SequenceDescription of Artificial Sequence Synthetic DNA 24 ccgctcgaggaggcctcttc cgctcccggc c 31 25 35 DNA Artificial Sequence Description ofArtificial Sequence Synthetic DNA 25 ccgaattcct attacggtgc tgtcaccacgctctg 35 26 31 DNA Artificial Sequence Description of ArtificialSequence Synthetic DNA 26 ccgctcgagg accctatgtg gactttgctg a 31 27 31DNA Artificial Sequence Description of Artificial Sequence Synthetic DNA27 ccgctcgagg atatgtggac tttgctgagt t 31 28 31 DNA Artificial SequenceDescription of Artificial Sequence Synthetic DNA 28 ccgctcgaggagtggacttt gctgagtttt a 31 29 31 DNA Artificial Sequence Description ofArtificial Sequence Synthetic DNA 29 ccgctcgagg agactttgct gagttttacc g31 30 31 DNA Artificial Sequence Description of Artificial SequenceSynthetic DNA 30 ccgctcgagg atttgctgag ttttaccgcc t 31 31 31 DNAArtificial Sequence Description of Artificial Sequence Synthetic DNA 31ccgctcgagg agctgagttt taccgcctct g 31 32 38 DNA Artificial SequenceDescription of Artificial Sequence Synthetic DNA 32 ccgaattcctattagaggcg gtaaaactca gcaaagtc 38 33 35 DNA Artificial SequenceDescription of Artificial Sequence Synthetic DNA 33 ccgaattcctattaagcaaa gtccacatag ggctc 35 34 32 DNA Artificial Sequence Descriptionof Artificial Sequence Synthetic DNA 34 ccgaattcct attaaaagtc cacatagggctc 32 35 32 DNA Artificial Sequence Description of Artificial SequenceSynthetic DNA 35 ccgaattcct attagtccac atagggctca ac 32 36 32 DNAArtificial Sequence Description of Artificial Sequence Synthetic DNA 36ccgaattcct attacacata gggctcaaca cg 32 37 32 DNA Artificial SequenceDescription of Artificial Sequence Synthetic DNA 37 ccgaattcctattaataggg ctcaacacga cc 32 38 32 DNA Artificial Sequence Description ofArtificial Sequence Synthetic DNA 38 ccgaattcct attagggctc aacacgaccg tc32 39 32 DNA Artificial Sequence Description of Artificial SequenceSynthetic DNA 39 ccgaattcct attactcaac acgaccgtcc tc 32 40 16 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide40 Cys Val Glu Pro Tyr Val Asp Phe Ala Glu Phe Tyr Arg Gly Arg Lys 1 510 15 41 16 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 41 Cys Gln Ser Pro Thr Leu Thr Leu Gln Ser Thr Asn ThrHis Thr Gln 1 5 10 15 42 1568 DNA Homo sapiens CDS (11)..(1549) 42gaattccacc atg gac tac aag gat gac gac gac aag atg gcg gcg cag 49 MetAsp Tyr Lys Asp Asp Asp Asp Lys Met Ala Ala Gln 1 5 10 agg agg agc ttgctg cag agt gag cag cag cca agc tgg aca gat gac 97 Arg Arg Ser Leu LeuGln Ser Glu Gln Gln Pro Ser Trp Thr Asp Asp 15 20 25 ctg cct ctc tgc cacctc tct ggg gtt ggc tca gcc tcc aac cgc agc 145 Leu Pro Leu Cys His LeuSer Gly Val Gly Ser Ala Ser Asn Arg Ser 30 35 40 45 tac tct gct gat ggcaag ggc act gag agc cac ccg cca gag gac agc 193 Tyr Ser Ala Asp Gly LysGly Thr Glu Ser His Pro Pro Glu Asp Ser 50 55 60 tgg ctc aag ttc agg agtgag aac aac tgc ttc ctg tat ggg gtc ttc 241 Trp Leu Lys Phe Arg Ser GluAsn Asn Cys Phe Leu Tyr Gly Val Phe 65 70 75 aac ggc tat gat ggc aac cgagtg acc aac ttc gtg gcc cag cgg ctg 289 Asn Gly Tyr Asp Gly Asn Arg ValThr Asn Phe Val Ala Gln Arg Leu 80 85 90 tcc gca gag ctc ctg ctg ggc cagctg aat gcc gag cac gcc gag gcc 337 Ser Ala Glu Leu Leu Leu Gly Gln LeuAsn Ala Glu His Ala Glu Ala 95 100 105 gat gtg cgg cgt gtg ctg ctg caggcc ttc gat gtg gtg gag agg agc 385 Asp Val Arg Arg Val Leu Leu Gln AlaPhe Asp Val Val Glu Arg Ser 110 115 120 125 ttc ctg gag tcc att gac gacgcc ttg gct gag aag gca agc ctc cag 433 Phe Leu Glu Ser Ile Asp Asp AlaLeu Ala Glu Lys Ala Ser Leu Gln 130 135 140 tcg caa ttg cca gag gga gtccct cag cac cag ctg cct cct cag tat 481 Ser Gln Leu Pro Glu Gly Val ProGln His Gln Leu Pro Pro Gln Tyr 145 150 155 cag aag atc ctt gag aga ctcaag acg tta gag agg gaa att tcg gga 529 Gln Lys Ile Leu Glu Arg Leu LysThr Leu Glu Arg Glu Ile Ser Gly 160 165 170 ggg gcc atg gcc gtt gtg gcggtc ctt ctc aac aac aag ctc tac gtc 577 Gly Ala Met Ala Val Val Ala ValLeu Leu Asn Asn Lys Leu Tyr Val 175 180 185 gcc aat gtc ggt aca aac cgtgca ctt tta tgc aaa tcg aca gtg gat 625 Ala Asn Val Gly Thr Asn Arg AlaLeu Leu Cys Lys Ser Thr Val Asp 190 195 200 205 ggg ttg cag gtg aca cagctg aac gtg gac cac acc aca gag aac gag 673 Gly Leu Gln Val Thr Gln LeuAsn Val Asp His Thr Thr Glu Asn Glu 210 215 220 gat gag ctc ttc cgt ctttcg cag ctg ggc ttg gat gct gga aag atc 721 Asp Glu Leu Phe Arg Leu SerGln Leu Gly Leu Asp Ala Gly Lys Ile 225 230 235 aag cag gtg ggg atc atctgt ggg cag gag agc acc cgg cgg atc ggg 769 Lys Gln Val Gly Ile Ile CysGly Gln Glu Ser Thr Arg Arg Ile Gly 240 245 250 gat tac aag gtt aaa tatggc tac acg gac att gac ctt ctc agc gct 817 Asp Tyr Lys Val Lys Tyr GlyTyr Thr Asp Ile Asp Leu Leu Ser Ala 255 260 265 gcc aag tcc aaa cca atcatc gca gag cca gaa atc cat ggg gca cag 865 Ala Lys Ser Lys Pro Ile IleAla Glu Pro Glu Ile His Gly Ala Gln 270 275 280 285 ccg ctg gat ggg gtgacg ggc ttc ttg gtg ctg atg tcg gag ggg ttg 913 Pro Leu Asp Gly Val ThrGly Phe Leu Val Leu Met Ser Glu Gly Leu 290 295 300 tac aag gcc cta gaggca gcc cat ggg cct ggg cag gcc aac cag gag 961 Tyr Lys Ala Leu Glu AlaAla His Gly Pro Gly Gln Ala Asn Gln Glu 305 310 315 att gct gcg atg attgac act gag ttt gcc aag cag acc tcc ctg gac 1009 Ile Ala Ala Met Ile AspThr Glu Phe Ala Lys Gln Thr Ser Leu Asp 320 325 330 gca gtg gcc cag gccgtc gtg gac cgg gtg aag cgc atc cac agc gac 1057 Ala Val Ala Gln Ala ValVal Asp Arg Val Lys Arg Ile His Ser Asp 335 340 345 acc ttc gcc agt ggtggg gag cgt gcc agg ttc tgc ccc cgg cac gag 1105 Thr Phe Ala Ser Gly GlyGlu Arg Ala Arg Phe Cys Pro Arg His Glu 350 355 360 365 gac atg acc ctgcta gtg agg aac ttt ggc tac ccg ctg ggc caa atg 1153 Asp Met Thr Leu LeuVal Arg Asn Phe Gly Tyr Pro Leu Gly Gln Met 370 375 380 agc cag ccc acaccg agc cca gcc cca gct gca gga gga cga gtg tac 1201 Ser Gln Pro Thr ProSer Pro Ala Pro Ala Ala Gly Gly Arg Val Tyr 385 390 395 cct gtg tct gtgcca tac tcc agc gcc cag agc acc agc aag acc agc 1249 Pro Val Ser Val ProTyr Ser Ser Ala Gln Ser Thr Ser Lys Thr Ser 400 405 410 gtg acc ctc tccctt gtc atg ccc tcc cag ggc cag atg gtc aac ggg 1297 Val Thr Leu Ser LeuVal Met Pro Ser Gln Gly Gln Met Val Asn Gly 415 420 425 gct cac agt gcttcc acc ctg gac gaa gcc acc ccc acc ctc acc aac 1345 Ala His Ser Ala SerThr Leu Asp Glu Ala Thr Pro Thr Leu Thr Asn 430 435 440 445 caa agc ccgacc tta acc ctg cag tcc acc aac acg cac acg cag agc 1393 Gln Ser Pro ThrLeu Thr Leu Gln Ser Thr Asn Thr His Thr Gln Ser 450 455 460 agc agc tccagc tct gac gga ggc ctc ttc cgc tcc cgg ccc gcc cac 1441 Ser Ser Ser SerSer Asp Gly Gly Leu Phe Arg Ser Arg Pro Ala His 465 470 475 tcg ctc ccgcct ggc gag gac ggt cgt gtt gag ccc tat gtg gac ttt 1489 Ser Leu Pro ProGly Glu Asp Gly Arg Val Glu Pro Tyr Val Asp Phe 480 485 490 gct gag ttttac cgc ctc tgg agc gtg gac cat ggc gag cag agc gtg 1537 Ala Glu Phe TyrArg Leu Trp Ser Val Asp His Gly Glu Gln Ser Val 495 500 505 gtg aca gcaccg tgatgagcgg ccgcatcgt 1568 Val Thr Ala Pro 510 43 513 PRT Homosapiens 43 Met Asp Tyr Lys Asp Asp Asp Asp Lys Met Ala Ala Gln Arg ArgSer 1 5 10 15 Leu Leu Gln Ser Glu Gln Gln Pro Ser Trp Thr Asp Asp LeuPro Leu 20 25 30 Cys His Leu Ser Gly Val Gly Ser Ala Ser Asn Arg Ser TyrSer Ala 35 40 45 Asp Gly Lys Gly Thr Glu Ser His Pro Pro Glu Asp Ser TrpLeu Lys 50 55 60 Phe Arg Ser Glu Asn Asn Cys Phe Leu Tyr Gly Val Phe AsnGly Tyr 65 70 75 80 Asp Gly Asn Arg Val Thr Asn Phe Val Ala Gln Arg LeuSer Ala Glu 85 90 95 Leu Leu Leu Gly Gln Leu Asn Ala Glu His Ala Glu AlaAsp Val Arg 100 105 110 Arg Val Leu Leu Gln Ala Phe Asp Val Val Glu ArgSer Phe Leu Glu 115 120 125 Ser Ile Asp Asp Ala Leu Ala Glu Lys Ala SerLeu Gln Ser Gln Leu 130 135 140 Pro Glu Gly Val Pro Gln His Gln Leu ProPro Gln Tyr Gln Lys Ile 145 150 155 160 Leu Glu Arg Leu Lys Thr Leu GluArg Glu Ile Ser Gly Gly Ala Met 165 170 175 Ala Val Val Ala Val Leu LeuAsn Asn Lys Leu Tyr Val Ala Asn Val 180 185 190 Gly Thr Asn Arg Ala LeuLeu Cys Lys Ser Thr Val Asp Gly Leu Gln 195 200 205 Val Thr Gln Leu AsnVal Asp His Thr Thr Glu Asn Glu Asp Glu Leu 210 215 220 Phe Arg Leu SerGln Leu Gly Leu Asp Ala Gly Lys Ile Lys Gln Val 225 230 235 240 Gly IleIle Cys Gly Gln Glu Ser Thr Arg Arg Ile Gly Asp Tyr Lys 245 250 255 ValLys Tyr Gly Tyr Thr Asp Ile Asp Leu Leu Ser Ala Ala Lys Ser 260 265 270Lys Pro Ile Ile Ala Glu Pro Glu Ile His Gly Ala Gln Pro Leu Asp 275 280285 Gly Val Thr Gly Phe Leu Val Leu Met Ser Glu Gly Leu Tyr Lys Ala 290295 300 Leu Glu Ala Ala His Gly Pro Gly Gln Ala Asn Gln Glu Ile Ala Ala305 310 315 320 Met Ile Asp Thr Glu Phe Ala Lys Gln Thr Ser Leu Asp AlaVal Ala 325 330 335 Gln Ala Val Val Asp Arg Val Lys Arg Ile His Ser AspThr Phe Ala 340 345 350 Ser Gly Gly Glu Arg Ala Arg Phe Cys Pro Arg HisGlu Asp Met Thr 355 360 365 Leu Leu Val Arg Asn Phe Gly Tyr Pro Leu GlyGln Met Ser Gln Pro 370 375 380 Thr Pro Ser Pro Ala Pro Ala Ala Gly GlyArg Val Tyr Pro Val Ser 385 390 395 400 Val Pro Tyr Ser Ser Ala Gln SerThr Ser Lys Thr Ser Val Thr Leu 405 410 415 Ser Leu Val Met Pro Ser GlnGly Gln Met Val Asn Gly Ala His Ser 420 425 430 Ala Ser Thr Leu Asp GluAla Thr Pro Thr Leu Thr Asn Gln Ser Pro 435 440 445 Thr Leu Thr Leu GlnSer Thr Asn Thr His Thr Gln Ser Ser Ser Ser 450 455 460 Ser Ser Asp GlyGly Leu Phe Arg Ser Arg Pro Ala His Ser Leu Pro 465 470 475 480 Pro GlyGlu Asp Gly Arg Val Glu Pro Tyr Val Asp Phe Ala Glu Phe 485 490 495 TyrArg Leu Trp Ser Val Asp His Gly Glu Gln Ser Val Val Thr Ala 500 505 510Pro 44 51 DNA Artificial Sequence Description of Artificial SequenceSynthetic DNA 44 cccgaattcc accatggact acaaggatga cgacgacaag atggcggcgca 51 45 35 DNA Artificial Sequence Description of Artificial SequenceSynthetic DNA 45 gatgcggccg ctcatcacgg tgctgtcacc acgct 35 46 34 DNAArtificial Sequence Description of Artificial Sequence Synthetic DNA 46ccgctcgagg acggcccgcc cactcgctcc cgcc 34 47 34 DNA Artificial SequenceDescription of Artificial Sequence Synthetic DNA 47 ccgctcgaggactcccgcct ggcgaggacg gtcg 34 48 34 DNA Artificial Sequence Descriptionof Artificial Sequence Synthetic DNA 48 ccgctcgagg agacggtcgt gttgagccctatgt 34

1. A method for screening substances that inhibit binding between a TAK1polypeptide and a TAB1 polypeptide, which method comprises contacting aTAB1 polypeptide to a TAK1 polypeptide and a test sample, and thendetecting or determining the TAK1 polypeptide that is bound to the TAB1polypeptide.
 2. A method for screening substances that inhibit bindingbetween the TAK1 polypeptide and the TAB1 polypeptide, which methodcomprises contacting a TAK1 polypeptide to a TAB1 polypeptide and a testsample, and then detecting or determining the TAB1 polypeptide that isbound to the TAK1 polypeptide.
 3. The screening method according toclaim 1 or 2, wherein the TAB1 polypeptide has an amino acid sequencecomprising Met at amino acid position 1 to Pro at amino acid position504 of the amino acid sequence as set forth in SEQ ID NO: 2 or having anamino acid sequence modified by the substitution, deletion and/oraddition of one or a plurality of amino acid residues of the amino acidsequence as set forth in SEQ ID NO: 2 and maintaining the biologicalactivity of the TAB1 polypeptide.
 4. The screening method according toclaim 1 or 2, wherein the TAK1 polypeptide has an amino acid sequencecomprising Met at amino acid position 1 to Ser at amino acid position579 of the amino acid sequence as set forth in SEQ ID NO: 4 or having anamino acid sequence modified by the substitution, deletion and/oraddition of one or a plurality of amino acid residues of the amino acidsequence as set forth in SEQ ID NO: 4 and maintaining the biologicalactivity of the TAK1 polypeptide.
 5. The screening method according toany of claims 1 to 4, wherein the TAB1 polypeptide is fused to anotherpeptide or polypeptide.
 6. The screening method according to any ofclaims 1 to 4, wherein the TAK1 polypeptide is fused to another peptideor polypeptide.
 7. The screening method according to any of claims 3 to6, wherein the TABI polypeptide is bound to a support.
 8. The screeningmethod according to any of claims 2 to 6, wherein the TAK1 polypeptideis bound to a support.
 9. The screening method according to claim 7 or 8wherein the support is beads or a plate.
 10. The screening methodaccording to claims 1 to 6 wherein the TAK1 polypeptide, the TAB1polypeptide, and the test sample are contacted under a homogeneouscondition.
 11. The screening method according to any of claim 1 andclaims 3 to 10, wherein the TAK1 polypeptide is a labeled TAK1polypeptide and said label is detected or determined.
 12. The screeningmethod according to any of claims 2 to 10, wherein the TAB1 polypeptideis a labeled TAB1 polypeptide and said label is detected or determined.13. The screening method according to claim 11 or 12 wherein the labeledTAK1 polypeptide or the labeled TAB1 polypeptide is labeled with aradioisotope, an enzyme or a fluorescent substance.
 14. The screeningmethod according to any of claims 3 to 10, wherein the TAK1 polypeptidethat is bound to the TAB1 polypeptide is detected or determined by aprimary antibody against the TAK1 polypeptide or a primary antibodyagainst another peptide or polypeptide that is fused to the TAK1polypeptide.
 15. The screening method according to any of claims 2 to10, wherein the TAK1 polypeptide that is bound to the TAB1 polypeptideis detected or determined by a primary antibody against the TAK1polypeptide or a primary antibody against another peptide or polypeptidethat is fused to the TAK1 polypeptide, and a secondary antibody againstthe primary antibody.
 16. The screening method according to any ofclaims 3 to 10, wherein the TAB1 polypeptide that is bound to the TAK1polypeptide is detected or determined by a primary antibody against theTAB1 polypeptide or a primary antibody against another peptide orpolypeptide, that is fused to the TAB1 polypeptide.
 17. The screeningmethod according to any of claims 2 to 10, wherein the TAB1 polypeptidethat is bound to the TAK1 polypeptide is detected or determined by aprimary antibody against the TAB1 polypeptide or a primary antibodyagainst another peptide or polypeptide that is fused to the TAB1polypeptide, and a secondary antibody against the primary antibody. 18.The screening method according to any of claims 14 to 17, wherein theprimary antibody or secondary antibody is labeled with a radioisotope,an enzyme or a fluorescent substance.
 19. A substance obtained by thescreening method according to any of claims 1 to
 18. 20. A substancethat inhibits binding between a TAK1 polypeptide and a TAB1 polypeptideobtained by a screening method according to any of claims 1 to
 18. 21.An inhibitor of signal transduction of TGF-β comprising a substance thatinhibits binding between a TAK1 polypeptide and a TAB1 polypeptideobtained by a screening method according to any of claims 1 to
 18. 22.An activator of signal transduction of TGF-β comprising a substance thatinhibits binding between a TAK1 polypeptide and a TAB1 polypeptideobtained by a screening method according to any of claims 1 to
 18. 23. Asuppressor of the enhanced production of extracellular matrix proteincomprising as. an active ingredient a substance that inhibits bindingbetween a TAK1 polypeptide and a TAB1 polypeptide obtained by ascreening method according to any of claims 1 to
 18. 24. An activator ofthe enhanced production of extracellular matrix protein comprising as anactive ingredient a substance that inhibits binding between a TAK1polypeptide and a TAB1 polypeptide obtained by a screening methodaccording to any of claims 1 to
 18. 25. A suppressor of the inhibitionof cellular growth comprising as an active ingredient a substance thatinhibits binding between a TAK1 polypeptide and a TAB1 polypeptideobtained by a screening method according to any of claims 1 to
 18. 26.An activator of the inhibition of cellular growth comprising as anactive ingredient a substance that inhibits binding between a TAK1polypeptide and a TAB1 polypeptide obtained by a screening methodaccording to any of claims 1 to
 18. 27. A suppressor of monocytemigration comprising as an active ingredient a substance that inhibitsbinding between a TAK1 polypeptide and a TAB1 polypeptide obtained by ascreening method according to any of claims 1 to
 18. 28. An activator ofmonocyte migration comprising as an active ingredient a substance thatinhibits binding between a TAK1 polypeptide and a TAB1 polypeptideobtained by a screening method according to any of claims 1 to
 8. 29. Asuppressor of the induction of biologically active substances comprisingas an active ingredient a substance that inhibits binding between a TAK1polypeptide and a TAB1 polypeptide obtained by the screening methodaccording to any of claims 1 to
 18. 30. An activator of the induction ofbiologically active substances comprising as an active ingredient asubstance that inhibits binding between a TAK1 polypeptide and a TAB1polypeptide obtained by a screening method according to any of claims 1to
 18. 31. A suppressor of immunosuppressive action comprising as anactive ingredient a substance that inhibits binding between a TAK1polypeptide and a TAB1 polypeptide obtained by a screening methodaccording to any of claims 1 to
 18. 32. An activator ofimmunosuppressive action comprising as an active ingredient a substancethat inhibits binding between a TAK1 polypeptide and a TAB1 polypeptideobtained by a screening method according to any of claims 1 to
 18. 33. Asuppressor of the deposition of amyloid β protein comprising as anactive ingredient a substance that inhibits binding between a TAK1polypeptide and a TAB1 polypeptide obtained by a screening methodaccording to any of claims 1 to
 18. 34. An activator of the depositionof amyloid β protein comprising as an active ingredient a substance thatinhibits binding between a TAK1 polypeptide and a TAB1 polypeptideobtained by a screening method according to any of claims 1 to
 18. 35. Asubstance that inhibits the function of a TAK1 polypeptide obtained by ascreening method according to any of claims 1 to
 18. 36. The substanceaccording to claim 35 in which the function is a kinase activity.